Methods and compounds modifying mitochondrial function

ABSTRACT

The invention provides methods for identifying modulators of mitochondrial function for therapeutic use in neurodegenerative disorder. The invention provides method for identifying subjects who may benefit from the therapeutic agents. Aspects of the methods include administering MIRO1 reducer to a subject having Parkinson&#39;s Disease. Also provided are companion diagnostic assays to determine if a subject is suitable for treatment with a MIRO1 reducer, and treating the subject in accordance with the results.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No.62/896,450, filed Sep. 5, 2019, which is incorporated herein in itsentirety for all purposes.

BACKGROUND

Neurons are metabolically active cells with high energy demands atlocations distant from the cell body. As a result, these cells areparticularly dependent on mitochondrial function, as reflected by theobservation that diseases of mitochondrial dysfunction often have aneurodegenerative component. Recent discoveries have highlighted thatneurons are reliant particularly on the dynamic properties ofmitochondria. Mitochondria are dynamic organelles by several criteria.They engage in repeated cycles of fusion and fission, which serve tointermix the lipids and contents of a population of mitochondria. Inaddition, mitochondria are actively recruited to subcellular sites, suchas the axonal and dendritic processes of neurons. Finally, the qualityof a mitochondrial population is maintained through mitophagy, a form ofautophagy in which defective mitochondria are selectively degraded.Defects in the key features of mitochondrial dynamics, such asmitochondrial fusion, fission, transport and mitophagy are associatedwith neurodegenerative disorder. Several major neurodegenerativedisorders—including Parkinson's, Alzheimer's and Huntington'sdisease—involve disruption of mitochondrial dynamics.

Mitochondrial movements are tightly controlled to maintain energyhomeostasis and prevent oxidative stress. Mitochondrial motility ceasesprior to the initiation of mitophagy, a crucial cellular mechanism bywhich depolarized mitochondria are degraded through autophagosomes andlysosomes. The arrest of motility may sequester damaged mitochondria,preventing them from moving and from reintroducing damage to otherhealthy mitochondria.

Miro is an outer mitochondrial membrane (OMM) protein that anchors themicrotubule motors kinesin and dynein to mitochondria (Glater E E,Megeath L J, Stowers R S, Schwarz T L. Axonal transport of mitochondriarequires milton to recruit kinesin heavy chain and is light chainindependent. The Journal of cell biology. 2006; 173:545-557;Koutsopoulos O S, Laine D, Osellame L, Chudakov D M, Parton R G, FrazierA E, Ryan M T. Human Miltons associate with mitochondria and inducemicrotubule-dependent remodeling of mitochondrial networks. Biochimicaet biophysica acta. 2010; 1803:564-574.). This depolarization-triggeredmitochondrial arrest is achieved by removal of Miro from the damagedmitochondrial surface (Wang X, Winter D, Ashrafi G, Schlehe J, Wong Y L,Selkoe D, Rice S, Steen J, LaVoie M J, Schwarz T L. PINK1 and Parkintarget Miro for phosphorylation and degradation to arrest mitochondrialmotility. Cell. 2011; 147:893-906.). Miro is subsequently degraded byproteasomes (Wang X, Winter D, Ashrafi G, Schlehe J, Wong Y L, Selkoe D,Rice S, Steen J, LaVoie M J, Schwarz T L. PINK1 and Parkin target Mirofor phosphorylation and degradation to arrest mitochondrial motility.Cell. 2011; 147:893-906.). Evidence has shown that two PD-linkedproteins, PINK1 (PTEN-induced putative kinase 1) and Parkin, act inconcert to target Miro for degradation (Ashrafi G, Schlehe J S, LaVoie MJ, Schwarz T L. Mitophagy of damaged mitochondria occurs locally indistal neuronal axons and requires PINK1 and Parkin. The Journal of cellbiology. 2014; 206:655-670.; Liu S, Sawada T, Lee S, Yu W, Silverio G,Alapatt P, Millan I, Shen A, Saxton W, Kanao T, et al. Parkinson'sdisease-associated kinase PINK1 regulates Miro protein level and axonaltransport of mitochondria. PLoS genetics. 2012; 8:e1002537; Wang X,Winter D, Ashrafi G, Schlehe J, Wong Y L, Selkoe D, Rice S, Steen J,LaVoie M J, Schwarz T L. PINK1 and Parkin target Miro forphosphorylation and degradation to arrest mitochondrial motility. Cell.2011; 147:893-906.). Mutations in PINK1 or Parkin are tied to rare formsof recessive early-onset PD.

Altered mitochondrial transport is one of the pathogenic changes inmajor adult-onset neurodegenerative diseases. In mutant LRRK2GS2019cells, the mitochondrial outer membrane protein Miro is stabilized andremains on damaged mitochondria for longer than normal, prolongingactive transport and inhibiting mitochondrial degradation (Hsieh et, al,2016). Miro degradation and mitochondrial motility are also impaired insporadic PD subjects. Prolonged retention of Miro, and the downstreamconsequences that ensue, may constitute a central component of PDpathogenesis.

Therefore, there is a need for novel therapeutic approaches and methodsto identify modulators of Miro retention and degradation that promotearrest of mitochondrial motility of damaged mitochondria and restorationof mitochondrial dynamics in neurodegenerative disease.

SUMMARY

Methods and compositions are provided for the monitoring, treatment, andscreening for a neurodegenerative disorder, e.g., Parkinson's Disease,in a subject. Aspects of the methods include administering MIRO1 reducerto a subject having Parkinson's Disease. Also provided are companiondiagnostic assays to determine if a subject is suitable for treatmentwith a MIRO1 reducer, and treating the subject in accordance with theresults.

In one aspect, provided is a method to determine the MIRO1 status of asubject comprising measuring the Miro1 response to mitochondrialdepolarization using biochemical assays, western blotting or ELISA, todetermine if a subject is deficient in the removal of MIRO1 followingdepolarization, wherein a subject deficient in the removal of MIRO1following depolarization is selected for treatment by administration ofa MIRO1 reducer. Determining the MIRO1 status can comprise detectingMIRO1 level in a subject, and comparing the MIRO1 level to a controlMIRO1 level in a control subject. In some embodiments, detecting MIRO1level comprises detecting MIRO1 in a tissue sample, such as a skinfibroblast, in the subject. In some embodiments, the method comprises anassay described in any one of the Examples.

Provided herein is a method to screen candidate agents for activity toreduce MIRO1 level, the method comprising: (a) contacting a candidateagent and a sample from a subject deficient in or suspected of beingdeficient in the removal of MIRO1, (b) measuring the MIRO1 level in thesample, (c) comparing the MIRO1 level in a sample with a control MIRO1level, and (d) assessing the activity of the candidate agent if theMIRO1 level in the sample is lower than the control MIRO1 level.

Provided here in a method of treating a neurodegenerative disordercomprising administering to a subject in need thereof a Miro-reducingagent, wherein the Miro-reducing agent reduces Miro in sporadicParkinson's disease cells with depolarized mitochondria by threestandard deviations or more as compared to reduction of Miro in controldepolarized sporadic Parkinson's disease cells with depolarizedmitochondria not contacted with the Miro-reducing agent. The healthycells can be healthy cells with depolarized mitochondria, wherein thehealthy cells are contacted with a mitochondrial depolarizing agent.

Provided herein is a method of treating a neurodegenerative disordercomprising administering to a subject in need thereof a Miro-reducingagent, wherein the Miro-reducing agent reduces Miro in Parkinson'sdisease cells with depolarized mitochondria by three standard deviationsor more as compared to reduction of Miro in control depolarized sporadicParkinson's disease cells with depolarized mitochondria not contactedwith the Miro-reducing agent.

Provided herein is a method of treating a neurodegenerative disordercomprising administering to a subject in need thereof a Miro-reducingagent, wherein: (a) the Miro-reducing agent reduces Miro in Parkinson'sdisease cells with depolarized mitochondria by two standard deviationsor more as compared to reduction of Miro in control Parkinson's diseasecells with depolarized mitochondria not contacted with the Miro-reducingagent; and (b) the Miro 1 reducing agent reduces Miro in Parkinson'sdisease cells with non-depolarized mitochondria by one standarddeviation or less as compared to reduction of Miro in controlParkinson's disease cells with non-depolarized mitochondria notcontacted with the Miro-reducing agent.

The Parkinson's disease cells can be Parkinson's disease cells withdepolarized mitochondria contacted with a mitochondrial depolarizingagent. The Parkinson's disease cells can be sporadic Parkinson's diseasecells. The Parkinson's disease cells can be fibroblasts.

Provided herein is a method of treating a neurodegenerative disordercomprising administering to a subject in need thereof a Miro-reducingagent, wherein the Miro-reducing agent reduces Miro in the followingassay: (a) fibroblast cells from a sporadic Parkinson's disease patientare plated into wells of an array (b) 24 hours after step (a), acandidate agent is added to a test well and the candidate agent is notadded to a control well; (c) 10 hours after step (b), FCCP is added tothe test well and control well; (d) 14 hours after step (c) cells of thetest well and control well are fixed with ice-cold 90% methanol; and (e)the cells of the test well and control well are immunostained withanti-Miro and 4′,6-diamidino-2-phenylindole, dihydrochloride (DAPI) andimaged with a confocal microscope to measure Miro intensity/cell forthose images of the test well and control well; wherein when thecandidate agent reduces Miro in the test well by three standarddeviations or more relative to the control well, the candidate agent isa Miro-reducing agent.

Provided herein is a method of treating a neurodegenerative disordercomprising administering to a subject in need thereof a Miro-reducingagent, wherein the Miro-reducing agent reduces Miro in sporadicParkinson's disease cells according to the following assay: (a)fibroblast cells from a sporadic Parkinson's disease patient are platedinto wells of an array; (b) 24 hours after step (a), a candidate agentis added to a first test well and the candidate agent is not added to afirst control well; (c) 10 hours after step (b), FCCP is added to afirst test well and first control well; (d) 14 hours after step (c),cells of the first test well and first control well are fixed withice-cold 90% methanol; and (e) the cells of the first test well andfirst control well are immunostained with anti-Miro and4′,6-diamidino-2-phenylindole, dihydrochloride (DAPI) and imaged with aconfocal microscope to measure Miro intensity/cell for those images ofthe first test well and first control well; and wherein theMiro-reducing agent does not reduce Miro in sporadic Parkinson's diseasecells according to the following assay: (a1) fibroblast cells from asporadic Parkinson's disease patient are plated into wells of an array;(b1) 24 hours after step (a1), the candidate agent is added to a secondtest well and is not added to a second control well; (c1) 14 hours afterstep (b1), cells of the second test well and second control well arefixed with ice-cold 90% methanol; and (d1) the cells of the second testwell and second control well are immunostained with anti-Miro and4′,6-diamidino-2-phenylindole, dihydrochloride (DAPI) and imaged with aconfocal microscope to measure Miro intensity/cell for those images ofthe second test well and second control well; wherein when the candidateagent reduces Miro in the first test well by two standard deviations ormore relative to the first control well, and the candidate agent reducesMiro in the second test well by less than one standard deviationrelative to the second control well, the candidate agent is aMiro-reducing agent.

In certain embodiments, the neurodegenerative disorder is Parkinson'sDisease (PD). In some such embodiments, the PD is a familial form of PD,e.g. a PTEN-induced putative kinase 1 (PINK-1)-associated form of PD, aParkin-associated form of PD, an LRRK2-associated form of PD, analpha-Synuclein (SNCA)-associated form of PD, an E3 ligase(Parkin)-associated PD, a GBA-associated PD, a ubiquitincarboxy-terminal hydrolase L1 (UCHL1)-associated form of PD, a parkinsonprotein 7 (PARK7, DJ-1) associated form of PD, an ATP13A2-associatedform of PD, a phospholipase A2, group VI (PLA2G6) -associated form ofPD, a DnaJ (Hsp40) homolog, subfamily C, member 6 (DNAJC6,PARK19)-associated form of PD; a eukaryotic translation initiationfactor 4 gamma, 1 (EIF4G1, PARK18)-associated form of PD; a F-boxprotein 7 (FBXO7)-associated form of PD; a GRB10 interacting GYF protein2 (GIGYF2)-associated form of PD; a HtrA serine peptidase 2(HTRA2)-associated form of PD; a synaptojanin 1 (SYNJ1)-associated formof PD; and a vacuolar protein sorting 35 homolog (VPS35)-associated formof PD. In other such embodiments, the PD is a sporadic form ofParkinson's Disease, for example, it is associated with a sporadicmutation in one of the aforementioned genes. In certain suchembodiments, the MIRO1 reducer is administered to the midbrain and/orputamen of the subject.

In some embodiments, an assay is provided for determining the MIRO1status of a subject. In some embodiments an assay is performed on apopulation of patient cells, conveniently fibroblasts are used, toidentify the MIRO1 phenotype of a subject. Miro1 is localized on themitochondrial surface and mediates mitochondrial motility. Miro1 isremoved from depolarized mitochondria to facilitate their clearance viamitophagy. Measuring the Miro1 response to mitochondrial depolarizationusing biochemical assays, ELISA, etc. shows that a high percentage ofParkinson's Disease subjects are deficient in the removal of MIRO1following depolarization.

As described in the Examples herein, MIRO1 status correlates with thepresence of certain neurodegenerative disorders. For instance, anelevated level of MIRO1 is associated with Parkinson's disease.

The Miro-reducing agent can reduce intracellular calcium levels by 20%or more. The Miro-reducing agent can have a molecular weight of from 100to 2000 Daltons. The Miro-reducing agent can be an antibody, peptide, orprotein or portion of one thereof. The Miro-reducing agent can bind toan EF hand protein. The Miro-reducing agent can be a calcium channelblocker. The calcium channel blocker can be an L-type and N-type calciumchannel blocker. The subject can have elevated Miro. The subject can beotherwise asymptomatic for the neurodegenerative disorder. Theneurodegenerative disorder can be Parkinson's disease.

Provided herein is a method for selecting a subject for treatment with atherapeutic agent for a neurodegenerative disorder, comprising: (a)collecting cells from the subject and evaluating a first control portionof the cells for the pre-depolarization Miro level in the cells; (b)contacting a second test portion of the cells with a depolarizing agent;and (c) evaluating the post-depolarization Miro level in the second testportion of cells contacted with the depolarizing agent and comparing theMiro level to the pre-depolarization Miro level in the first controlportion of cells; wherein when the post-depolarization Miro level in thesecond test portion of cells is reduced by 40% or less relative to thepre-depolarization Miro level in the first control portion of cells, thesubject is treated with a therapeutic agent for neurodegenerativedisorder. Provided herein is a method for selecting a subject fortreatment with a therapeutic agent for a neurodegenerative disorder,wherein when the post-depolarization Miro level in the second testportion of cells is reduced by 10% to 50% relative to thepre-depolarization Miro level in the first control portion of cells, thesubject is treated with a therapeutic agent for neurodegenerativedisorder.

The neurodegenerative disorder can be Parkinson's disease. The subjectcan be asymptomatic for Parkinson's disease. The therapeutic agent canbe selected from levodopa and dopamine antagonists. The therapeuticagent can be a Miro-reducing agent. The therapeutic agent can have amolecular weight of from 100 to 2000 Daltons. The therapeutic agent canan antibody, peptide, or protein or portion of one thereof. Thetherapeutic agent can bind to an EF hand protein. The therapeutic agentcan be a calcium channel blocker. The calcium channel blocker can be anL-type and N-type calcium channel blocker.

Provided herein is a method of improving a condition of aging,comprising administering to a subject in need thereof a Miro-reducingagent. The condition of aging can be selected from memory impairment,muscle degeneration, arthritis, cardiovascular disease, osteoporosis,glaucoma, dementia, macular degeneration, and cataract. TheMiro-reducing agent can be administered prophylactically.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

Before the present methods and compositions are described, it is to beunderstood that aspects of the invention are not limited to particularmethod or composition described, as such may, of course, vary. It isalso to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting, since the scope of the present invention will be limitedonly by the appended claims.

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIGS. 1A-1F. Miro1 Response to CCCP in Fibroblasts. FIG. 1A: Schematicrepresentation of our readouts. FIGS. 1B-1C: Examples of the readoutsusing healthy-1, PD-2, and risk-2. FIG. 1D: Heat maps show the relativemitochondrial protein levels. The intensity of each band in themitochondrial fraction is normalized to that of the mitochondrialloading control VDAC from the same blot and expressed as a fraction ofthe mean of Healthy-1 with DMSO treatment; this control was included inevery experiment. Mean values are imported into heat maps. n=3-35. FIGS.1E-1F: ELISA of Miro1. n=4 with duplicates each time. Comparison withinthe same subject. Throughout *: P<0.05; **: P<0.01; ***: P<0.001.

FIGS. 2A-2H show Miro1 ratio (CCCP/DMSO) vs. different variables. FIG.2A: healthy subjects and Parkinson's disease subjects (“PD”); FIG. 2B:male and female subjects; FIG. 2C: age at sampling (years); FIG. 2D: PDonset age (years); FIG. 2E: PD progression (years with PD); FIG. 2F:UPDRS; FIG. 2G: Hoehn and Yahr scale of PD subjects; FIG. 2H:mini-mental state examination in PD subjects.

FIGS. 3A-3G: FIG. 3A shows relative β-actin levels or Miro1 levels(ng/ml) in subjects; FIG. 3B shows absorbance at 450 nm vs. β-actinlevels; FIG. 3C shows relative β-actin levels vs. capture antibody; FIG.3D shows absorbance at 450 nm vs. Miro1 levels; FIG. 3E shows relativeMiro1 levels vs. capture antibody; FIG. 3F shows absorbance at 450 nmvs. Miro1 (ng/ml); FIG. 3F shows Miro1 (ng/ml) vs. capture antibody.

FIGS. 4A-4F show a schematic representation of an in vitro customdesigned screen for Miro1 drug discovery. FIG. 4A depicts the culture offibroblast cells obtained from both healthy subjects and subjects withsporadic PD. FIG. 4B depicts the transfer of fibroblasts cells byseeding fibroblast cells into 384-well plates. FIG. 4C shows the step ofaddition of compound libraries to fibroblasts in 384-well plates. FIG.4D shows the performance of immunocytochemistry (ICC) on the fibroblastcells treated with compound libraries. FIG. 4E shows the procedure fordetection of Miro1 protein levels under the confocal microscope andexemplary images of DAPI and Miro1 in one individual well. FIG. 4F showsthe custom data analysis algorithm pipeline designed to identifycompounds altering Miro1 levels following mitochondrial depolarization.

DETAILED DESCRIPTION

In one aspect, the disclosure herein provides a method of diagnosing aneurodegenerative disorder, e.g., Parkinson's disease, comprisingdetecting Miro, e.g., Miro1, in a subject. In certain embodiments, theMiro1 level in a subject, e.g., Miro1 level in a tissue sample from asubject, is detected using an assay described herein. The Miro1 level ina subject can be compared to a control Miro1 level in a control subjectin order to determine the presence or absence of a neurodegenerativedisorder. For example, a Miro1 level that is higher, such as 20% ormore, or 30% or more, than a control Miro1 level, it can indicate thepresence of Parkinson's disease. If the presence of a neurodegenerativedisorder is indicated, the subject can be treated for theneurodegenerative disorder, such as with a treatment known in the art ordescribed herein.

In one aspect, the disclosure herein provides a method of treating aneurodegenerative disorder, e.g., Parkinson's disease, comprisingadministering to a subject in need thereof a Miro-reducing agent, e.g.,a Miro1-reducing agent. In certain embodiments, the Miro-reducing agentis identified using an assay described herein. In certain embodiments,the Miro-reducing agent reduces Miro, e.g., Miro1, levels in cells, forexample, cells from a subject deficient in or suspected of beingdeficient in the removal of Miro 1. In certain embodiments, theMiro-reducing agent reduces Miro, e.g., Miro1, levels in cells to alevel consistent with Miro, e.g., Miro1, levels in control cells.

In certain embodiments, the disclosure provides methods of treating asubject with a neurodegenerative disorder, e.g., Parkinson's disease,wherein the subject is diagnosed with Parkinson's disease using a methoddescribed herein. Methods of treating neurodegenerative disorder withMiro-reducing agents are described below.

In certain embodiments, the disclosure provides a method of identifyingMiro1 reducing agents. In certain embodiments, the disclosure provides amethod of screening candidate agents for use in the treatment of aneurodegenerative disorder, e.g., Parkinson's disease, using an assaydescribed herein.

Before the present invention is further described, it is to beunderstood that this invention is not limited to particular embodimentsdescribed, as such may, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting, sincethe scope of the present invention will be limited only by the appendedclaims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges, and are also encompassed within the invention, subjectto any specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, the preferredmethods and materials are now described. All publications mentionedherein are incorporated herein by reference to disclose and describe themethods and/or materials in connection with which the publications arecited.

The terms “treatment”, “treating” and the like are used herein togenerally mean obtaining a desired pharmacologic and/or physiologiceffect. The effect may be prophylactic in terms of completely orpartially preventing a disease or symptom thereof and/or may betherapeutic in terms of a partial or complete cure for a disease and/oradverse effect attributable to the disease. “Treatment” as used hereincovers any treatment of a disease in a mammal, and includes: (a)preventing the disease from occurring in a subject which may bepredisposed to the disease but has not yet been diagnosed as having it;(b) inhibiting the disease, i.e., arresting its development; or (c)relieving the disease, i.e., causing regression of the disease. Thetherapeutic agent may be administered before, during or after the onsetof disease or injury. The treatment of ongoing disease, where thetreatment stabilizes or reduces the undesirable clinical symptoms of thesubject, is of particular interest. Such treatment is desirablyperformed prior to complete loss of function in the affected tissues.The subject therapy will desirably be administered during thesymptomatic stage of the disease, and in some cases after thesymptomatic stage of the disease.

The terms “individual,” “subject,” “host,” and “patient,” are usedinterchangeably herein and refer to any mammalia subject for whomdiagnosis, treatment, or therapy is desired, particularly humans.

It must be noted that as used herein and in the appended claims, thesingular forms “a,” “and,” and “the” include plural referents unless thecontext clearly dictates otherwise. It is further noted that the claimsmay be drafted to exclude any optional element. As such, this statementis intended to serve as antecedent basis for use of such exclusiveterminology as “solely,” “only” and the like in connection with therecitation of claim elements, or use of a “negative” limitation.

The terms “candidate agent,” “test agent,” “agent,” “substance,” and“compound” are used interchangeably herein. Candidate agents encompassnumerous chemical classes, typically synthetic, semi-synthetic, ornaturally-occurring inorganic or organic molecules. Candidate agentsinclude those found in large libraries of synthetic or naturalcompounds.

Mitochondrial Rho (Miro) is a small GTPase known for its functions inmitochondria transport and homeostasis. Miro is a highly conservedprotein across unicellular and multicellular eukaryotes. Miro1 and Miro2are conserved single-pass transmembrane integral proteins withN-terminal regions exposed to the cytosolic side and consist of twoGTPase domains separated by a pair of canonical EF hands. Miro alsocontains a C-terminal hydrophobic domain that allows membrane insertion.Miro GTPases are localized to the mitochondrial outer membrane and playcritical roles in intracellular mitochondria movement in metazoans,particularly over long distances along microtubule tracts in neurons.Miro is also known to play a role in mediating intercellular transportof mitochondria between cells via tunneling nanotubes. Miro mediatesbidirectional mitochondrial movement along microtubule tracts byengaging both kinesin and dynein. Miro engages with microtubule tracksvia the cargo adaptors of the Milton/Trak family. Miro also transportsmitochondria along the actin tracks using the mitochondrial actin motorMyo19. Miro is also involved in peroxisomal distribution through bindingwith Peroxin 26. In addition, Miro also has roles in mitochondrialfusion and fission dynamics through its modulation of the mitochondrialdynamin Drp1 and interactions with the mitochondrial fusion proteinsmitofusins 1 and 2.

Miro turnover is regulated by the PTEN-induced putative kinase 1(PINK1)/Parkin pathway. PINK1 phosphorylates Miro, thus promoting itsinteraction with the E3 ubiquitin ligase Parkin. Parkin promotes Miroubiquitination and degradation, which effectively arrests axonaltransport of damaged mitochondria. On the other hand, PINK1phosphorylated Miro also recruits Parkin to the damaged mitochondria,which then tags these for mitophagic destruction.

Miro is located in the outer mitochondrial membrane and anchors themicrotubule motors kinesin and dynein to mitochondria. Uponmitochondrial damage caused by mitochondrial depolarization, mitophagyis initiated in the cell. Mitochondrial motility ceases prior to theinitiation of mitophagy, a cellular mechanism, by which depolarizedmitochondria are degraded through autophagosomes and lysosomes. Thearrest of motility may sequester damaged mitochondira, preventing themfrom moving on and from reintroducing damage to other healthymitochondria. This depolarization-triggered mitochondrial arrest isachieved by removal of Miro from the damaged mitochondrial surface andsubsequent degradation by proteasomes. However, a notable impairment inthe process of Miro degradation and subsequent clearance of damagedmitochondria is observed in skin fibroblasts from both familial andsporadic PD subjects.

The EF hands of Miro mediate Ca²⁺-dependent arrest of bidirectionalmitochondrial transport. Mitochondrial transport is mediated by linkingMiro to KIF5. Ca²⁺ binding detaches KIF5 from mitochondria or by turning“off” KIF5 engagement with microtubules. Ca²⁺ binding to the EF handstriggers the direct interaction of the motor domain with Miro, thuspreventing the motor from engaging with MTs.

As used herein “Miro’ can refer to any one or all of the following:family members, isoforms, homologs, paralogs, mutants, alleles,variants, derivatives, fragments, species, coding and noncodingsequences, sense and antisense polynucleotide strands, etc. Miro refersto human sequence Miro, such as the complete amino acid sequence ofhuman Miro isoforms having Uniprot Identifiers Q8IX12-1, Q8IX12-2,Q8IX12-3, Q8IX12-4, Q8IX12-5, Q8IX12-6 or Q8IX12-7. The human Mirosequence may differ from human Miro isoforms of Uniprot identifiersQ8IX12-1, Q8IX12-2, Q8IX12-3, Q8IX12-4, Q8IX12-5, Q8IX12-6 orQ8IX12-7_(—) by having, for example, conserved mutations or mutations innon-conserved regions and the miro has substantially the same biologicalfunction as the human Miro isoforms of Q8IX12-1, Q8IX12-2, Q8IX12-3,Q8IX12-4, Q8IX12-5, Q8IX12-6 or Q8IX12-7 such as binding tomitochondria. Miro is also referred to as used RHOT1, and MitochondrialRho GTPase1. Miro includes isoforms of Miro such as Miro1 and Miro2.

A particular Miro sequence can be at least 90%, at least 95%, or even atleast 96%, at least 97%, at least 98%, or at least 99% identical inamino acid sequence to Miro of Uniprot identifiers Q8IXI2-1, Q8IXI2-2,Q8IXI2-3, Q8IXI2-4, Q8IXI2-5, Q8IXI2-6 or Q8IXI2-7. In certainembodiments, a human Miro sequence can differ by more than 10 amino aciddifferences from the Miro sequence of Uniprot identifiers Q8IXI2-1,Q8IXI2-2, Q8IXI2-3, Q8IXI2-4, Q8IXI2-5, Q8IXI2-6 or Q8IXI2-7. In certainembodiments, the human Miro may display no more than 5, or even no morethan 4, no more than 3, no more than 2, or no more than 1 amino aciddifference from the Miro sequence of Uniprot identifiers Q8IXI2-1,Q8IXI2-2, Q8IXI2-3, Q8IXI2-4, Q8IXI2-5, Q8IXI2-6 or Q8IXI2-7. Percentidentity can be determined as described herein.

An “isoform” of a protein can be, e.g., a protein resulting fromalternative splicing of a gene expressing the protein, or a degradationproduct of the protein. “Sequence homology”, refers to anucleotide-to-nucleotide or amino acid-to-amino acid correspondence oftwo polynucleotides or polypeptide sequences, respectively. As usedherein, “sequence identity” or “identity” refers, in the context of twonucleic acid sequences or amino acid sequences, to the residues in thetwo sequences that are the same when aligned for maximum correspondenceover a specified comparison window.

As used herein, “percent sequence identity” means the value determinedby comparing two optimally aligned sequences over a comparison window,wherein the portion of the polynucleotide or polypeptide sequence in thecomparison window may comprise additions or deletions, i.e., gaps,compared to the reference sequence which does not comprise additions ordeletions can be used for optimal alignment of the two sequences. Thepercentage can be calculated by: determining the number of positions atwhich the identical nucleotide or amino acid occurs in both sequences toyield the number of matched positions; dividing the number of matchedpositions by the total number of positions to give in the comparisonwindow; and multiplying the result by 100 to determine the percentage ofsequence identity.

Sequence comparisons, such as for the purpose of assessing identities,may be performed by any suitable alignment algorithm, including but notlimited to the Needleman-Wunsch algorithm (see, e.g., the EMBOSS Needlealigner available at www.ebi.ac.uk/Tools/psa/emboss_needle/, optionallywith default settings), the BLAST algorithm (see, e.g., the BLASTalignment tool available at blast.ncbi.nlm.nih.gov/Blast.cgi, optionallywith default settings), and the Smith-Waterman algorithm (see, e.g., theEMBOSS Water aligner available at www.ebi.ac.uk/Tools/psa/emboss_water/,optionally with default settings). Optimal alignment may be assessedusing any suitable parameters of a chosen algorithm, including defaultparameters.

The “percent identity” between two sequences may be calculated as thenumber of exact matches between two optimally aligned sequences dividedby the length of the reference sequence and multiplied by 100. Percentidentity may also be determined, for example, by comparing sequenceinformation using the advanced BLAST computer program, including version2.2.9, available from the National Institutes of Health. The BLASTprogram can be based on the alignment method of Karlin and Altschul,Proc. Natl. Acad. Sci. USA 87:2264-2268 (1990) and as discussed inAltschul, et al., J. Mol. Biol. 215:403-410 (1990); Karlin and Altschul,Proc. Natl. Acad. Sci. USA 90:5873-5877 (1993); and Altschul et al.,Nucleic Acids Res. 25:3389-3402 (1997). Briefly, the BLAST program candefine identity as the number of identical aligned symbols (i.e.,nucleotides or amino acids), divided by the total number of symbols inthe shorter of the two sequences. The program may be used to determinepercent identity over the entire length of the sequences being compared.Default parameters can be provided to optimize searches with short querysequences, for example, with the blastp program. The program can alsoallow use of an SEG filter to mask-off segments of the query sequencesas determined by the SEG program of Wootton and Federhen, Computers andChemistry 17: 149-163 (1993). High sequence identity can includesequence identity in ranges of sequence identity of approximately 80% to99% and integer values there between.

A “homolog” can refer to any sequence that has at least 50%, at least55%, at least 60%, at least 65%, at least 70%, at least 75%, at least80%, at least 85%, at least 90%, at least 95%, at least at least 96%, atleast 97%, at least 98%, at least 99%, or at least 99.5% sequencehomology to another sequence. In certain embodiments, a homolog has from70% to 99%, from 80% to 99%, from 85% to 99%, from 90% to 99% or evenfrom 95% to 99% sequence homology to another sequence. In some cases,the homolog can have a functional or structural equivalence with thenative or naturally occurring sequence. In some cases, the homolog canhave a functional or structural equivalence with a domain, a motif or apart of the protein, that is encoded by the native sequence or naturallyoccurring sequence.

Homology comparisons may be conducted with sequence comparison programs.Computer programs may calculate percent (%) homology between two or moresequences and may also calculate the sequence identity shared by two ormore amino acid or nucleic acid sequences. Sequence homologies may begenerated by any of a number of computer programs, for example BLAST orFASTA, etc. A suitable computer program for carrying out such analignment is the GCG Wisconsin Bestfit package (University of Wisconsin,U.S.A; Devereux et al., 1984, Nucleic Acids Research 12:387). Examplesof other software than may perform sequence comparisons include, but arenot limited to, the BLAST package (see Ausubel et al., 1999 ibid—Chapter18), FASTA (Atschul et al., 1990, J. Mol. Biol., 403-410) and theGENEWORKS suite of comparison tools. Both BLAST and FASTA are availablefor offline and online searching (see Ausubel et al., 1999 ibid, pages7-58 to 7-60).

Percent homology may be calculated over contiguous sequences, i.e., onesequence is aligned with the other sequence and each amino acid ornucleotide in one sequence is directly compared with the correspondingamino acid or nucleotide in the other sequence, one residue at a time.This is called an “ungapped” alignment. Typically, such ungappedalignments can be performed over a relatively short number of residues.

In an otherwise identical pair of sequences, one insertion or deletionmay cause the following amino acid or nucleotide residues to be put outof alignment, thus potentially resulting in a large reduction in %homology when a global alignment is performed. Consequently, thesequence comparison method can be designed to produce optimal alignmentsthat take into consideration possible insertions and deletions withoutunduly penalizing the overall homology or identity score. This can beachieved by inserting “gaps” in the sequence alignment to try tomaximize local homology or identity.

Calculation of maximum % homology can make use of optimal alignment,taking into consideration gap penalties. BLAST 2 Sequences is anothertool that can be used for comparing protein and nucleotide sequences(see FEMS Microbiol Lett. 1999 174(2): 247-50; FEMS Microbiol Lett. 1999177(1): 187-8 and the website of the National Center for Biotechnologyinformation at the website of the National Institutes for Health).

Homologous sequences can also have deletions, insertions orsubstitutions of amino acid residues which result in a functionallyequivalent substance. Deliberate amino acid substitutions may be made onthe basis of similarity in amino acid properties (such as polarity,charge, solubility, hydrophobicity, hydrophilicity, and/or theamphipathic nature of the residues) and it is therefore useful to groupamino acids together in functional groups. Amino acids may be groupedtogether based on the properties of their side chains alone. Sets ofconserved amino acids may be described in the form of a Venn diagram(Livingstone C. D. and Barton G. J. (1993) “Protein sequence alignments:a strategy for the hierarchical analysis of residue conservation”Comput. Appl. Biosci. 9: 745-756) (Taylor W. R. (1986) “Theclassification of amino acid conservation” J. Theor. Biol. 119;205-218).

“Miro-reducing agent”, e.g., a MIRO1 reducer, refers to any agent thatdecreases the level of a Miro protein, or a homolog thereof, in cellswith depolarized mitochondria. In an exemplary embodiment, aMiro-reducing agent may decrease at least one biological activity of aMiro protein in a cell with depolarized mitochondria. Exemplarybiological activities of Miro include promoting mitochondrial transport,mitophagy, microtubule binding, mitochondrial fission and fusion amongothers. A Miro-reducing agent can be, for example, a small molecule, apeptide, an aptamer, a protein or a functional fragment of a protein. Afunctional fragment of a protein, as used here, refers to all or part ofthe molecular elements of a protein which affect a specified functionsuch as protein binding, signal transduction etc.

In some embodiments, a MIRO1 reducer will inhibit the level orbiological activity of MIRO1 by 20% or more, for example, 30% or more,40% or more, or 50% or more, sometimes 60% or more, 70% or more, or 80%or more, e.g. 90%, 95%, or 100%, relative to an untreated control notcontacted with the reducer. A reducer may be validated as such by anyconvenient method in the art for detecting the level and/or activity ofMIRO1 in the presence versus absence of the MIRO1 reducer.

For example, the level and/or the phosphorylation state of a Miroprotein (Ser156, Thr298 or Thr299 of Miro1 and Miro2, see, e.g., Wang etal. Cell 2011, 147(4): 893-906) may be detected, for example byimmunoprecipitation with a mitochondrial transport protein-specificantibody followed by Western blotting with a phospho-specific or ageneral antibody, where an increase in phosphorylation of Miro proteinsand/or a decrease of total Miro protein levels, or a decrease inphosphorylation of Khc following contact with the agent may indicatethat the agent will treat Parkinson's Disease. As another example, thelevel and/or the ubiquitination of a Miro protein may be detected, forexample by immunoprecipitation with a mitochondrial transportprotein-specific antibody followed by Western blotting with aubiquitin-specific antibody, where an increase in ubiquitinationfollowing contact with the candidate agent indicates that the agent willtreat Parkinson's Disease. As another example, the ability of the targetmitochondrial protein to transport mitochondria within a cell may beassessed by, for example, treating cultured cells (e.g., neurons) withthe MIRO1 reducer and observing the transport of mitochondria in thecells as compared to cells not treating with the MIRO1 reducer, e.g.,using live cell imaging techniques (see, e.g., Brickley and StephensonJ. Biol Chem 286(20): 18079-92 (2011); Misko et al. J Neurosci 30(19):4232-40 (2010); Russo G J et al. J. Neurosci 29(17):5443-55 (2009)). Asanother example, because the formation of a complex between Miro (e.g.,Miro 1 and 2), TRAK (e.g., TRAK1 and 2), and Khc is essential formitochondrial transport in neurons (see e.g., Brickley and Stephenson J.Biol Chem 286(20): 18079-92 (2011)), the effect of MIRO1 reducer on Mirofunction may be assessed by assessing the ability of Miro, TRAK and Khcto form a complex in the presence of the MIRO1 reducer. Such anassessment can be performed using any technique to determineprotein-protein interaction including, but not limited to,co-immunoprecipitation and affinity purification techniques. In specificembodiments, the ability is assessed in a cell having a familial PDmutation, e.g. a PINK1 or LRRK2 mutation.

“Mitochondrial depolarization” is the process in which the membranepotential of the mitochondria changes in the depolarizing direction fromthe resting potential, from negative to positive. Normal, mild calciuminflux from cytosol into the mitochondrial matrix causes transientdepolarization that is corrected by pumping out protons. Chemicallyinduced mitochondrial depolarization provides a suitable assay fordetermining the effect of damage to mitochondria.

A number of agents are known and used experimentally to causemitochondrial depolarization and can be used in the methods of theinvention for that purpose. Such agents are generally in the class ofmitochondrial uncouplers or uncoupling agents that disrupt oxidativephosphorylation in mitochondria by dissociating the reactions of ATPsynthesis from the electron transport chain. The result is that themitochondrion expends energy to generate a proton motive force, but theproton motive force is dissipated before the ATP synthase can recapturethis energy and use it to make ATP.

The concentration of agent and time of exposure is sufficient touncouple or depolarize the mitochondria. For example, CCCP at aconcentration of from about 10-100 μM, for example from about 20 to 80μM, from about 30 to 50 μM, is sufficient. Dosage of other agents mayprovide for activity comparable to these concentrations of CCCP. Thecells are incubated for a period of time sufficient to depolarizemitochondria and initiate clearance, e.g. at least about 1 hour, atleast about 2 hours, and usually not more than about 24 hours, and maybe from about 1 to 24 hours, from about 2 to 20 hours, from about 3 to18 hours, from about 4 to 14 hours, from about 5 to 10 hours, and may befrom about 4 to 8 hours.

Carbonyl cyanide p-trifluoro-methoxyphenyl hydrazone (FCCP) andcarbonylcyanide-3-chlorophenylhydrazone (CCCP) are mitochondrialuncouplers frequently used in research. These molecules are lipophilicweak acids that act as protonophores. Due to their hydrophobic nature,these compounds can easily traffic across biological membranes and allowthe protons to cross these membranes. Other molecules suitable for thispurpose include 1,3-bis(3,5-dichlorophenyl)urea;dodecyltriphenylphosphonium; dinitrophenol; BAM15((2-fluorophenyl)6-[(2-fluorophenyl)amino](1,2,5-oxadiazolo[3,4-e]pyrazin-5-yl)amine),which is a mitochondria-specific protonophore uncoupler that possesses asimilar potency to FCCP or DNP; FR58P1 (a bromoalkyl ester of ahydroquinone derivative) is another mitochondrial protonophore.

Other mitochondria-specific uncouplers include MitoFluo, which is aconjugate of a triphenylphosphonium cation to fluorescein, acting as afluorescent uncoupler that accumulates preferentially in mitochondria;dodecyltriphenylphosphonium (C12TPP), which operates as a fatty acidanion carrier and facilitates fatty acid cycling across the membrane andthus mitochondria uncoupling; the Rhodamine 19 butyl ester C4R1, whichacts as a mild mitochondrial uncoupler; and MitoPhotoDNP, which is afusion of DNP, the o-nitrobenzyl group (a photoactivable group), andtriphenylphosphonium. Bupivacaine, a local anesthetic agent, can actpartially as a protonophore but also presents an inhibitory effect onstate 3-respiration by altering the mitochondrial proton pumpstoichiometry. Weak C-H acids, such as ortho-carborane (1,2-C₂B₁₀H₁₂),also have global uncoupling properties when used at concentrationscomparable to FCCP (10 μM range).

Affinity Assays, which are often immunoassays, are an assay or analyticprocedure that relies on the binding of the target molecule, i.e. Miro1,to receptors, antibodies or other macromolecules. A detection method isused to determine the presence and extent of the binding complexes thatare formed. Many formats for such assays are known and used in the art,and are suitable for detection of Miro1 degradation followingmitochondrial uncoupling or depolarization. In some embodiments, theassay format is suitable for high-throughput analysis.

Included in suitable assay formats are immunoassays that utilizeantibodies specific for Miro1. Suitable antibodies for this purpose areknown and commercially available as polycloncal or monoclonalcompositions, e.g. from Invitrogen, including monoclonals CL1095,CL1083; from Sigma Aldrich including clone 4H4, Santa Cruz BiotechnologyAnti-Rho T1 Antibody (A-8); and the like.

Assays of interest include, for example, Western blots;immunohistochemistry; immunoprecipitation; etc., and particularlyinclude immunoassays such as enzyme-linked immunosorbent assay (ELISA),radioimmunoassay (RIA); enzyme immunoassay (EIA).

Enzyme-linked immunosorbent assays (ELISAs) are used to qualitativelyand quantitatively analyze the presence or concentration of a particularsoluble antigen such as Miro1, in liquid samples, such as cell lysates.These assays generally make use of the ability of multiwell plates orothers to bind antibodies which trap the cognate antigen. Usually acolorimetric endpoint that can be detected via absorbance wavelength andquantitated from a known standard curve of antigen or antibody dilutionsis used. The detection antibody is often labelled with an enzyme such ashorseradish peroxidase or alkaline phosphatase, or a fluorescent tag, oran electrochemiluminescent label or through an intermediary label suchas biotin.

Common ELISA formats include the sandwich ELISA, so named because theanalyte is “sandwiched” between two different antibodies. The capturesubstrate in this format is a capture antibody, often a monoclonalantibody, to increase the specificity of the assay and reduce backgroundnoise. The analyte is bound to the capture antibody, then detected bybinding to a detection antibody. A variation of sandwich ELISA assay,called Single-Molecule Assay (Simoa), uses beads are coated with acapture antibody; each bead is bound to either one or zero targetmolecule, and individual beads are detected with another antibody(detection antibody) and a labeling enzyme.

Other ELISA formats include indirect ELISA, where the capture substrateis the specific antigen that is being tested and the detection step ismediated by a primary antibody and an enzyme-conjugated secondaryantibody which is reactive against the primary antibody. Thus, theprimary antibody that recognizes the antigen is not labeled. In a directELISA the capture substrate is the specific antigen that is beingtested, and the enzyme that catalyzes the color-change reaction isconjugated to the antigen detector antibody.

Immuno-PCR (I-PCR) is a technique that combines the sensitivity of thenucleic acid amplification by PCR with the specificity of theantibody-based assays resulting in an increase of the detectionsensitivity.

Methods of Screening

In some aspects of the invention, methods are provided for screeningcandidate agents for activity in treating Parkinson's Disease in asubject having Parkinson's Disease. To that end, it has been shown thatmitochondrial transport proteins, for example, Miro proteins,trafficking kinases, and the kinesin heavy chain, promote thedevelopment or progression of Parkinson's Disease or the symptomsthereof. Accordingly, screening for candidate agents that inhibit theexpression or activity of mitochondrial transport proteins in cellsshould identify agents that will be useful in treating Parkinson'sDisease in subjects. In particular agents are tested for the ability toreduce levels of Miro1 following mitochondrial depolarization, andpreferably without a substantial reduction in basal Miro1 levels.

Test agents can be obtained using any of the numerous approaches incombinatorial library methods known in the art, including: biologicallibraries; spatially addressable parallel solid phase or solution phaselibraries; synthetic library methods requiring deconvolution; the‘one-bead one-compound’ library method; and synthetic library methodsusing affinity chromatography selection.

In one embodiment, to identify agents that alter Miro1 degradation; acell, e.g. a viable cell or population of cells, is contacted with anagent to be tested; alternatively, the protein can be contacted directlywith the agent to be tested. The level (amount) of activity is assessedeither directly or indirectly, and is compared with the level ofactivity in a control, e.g. in the absence of the agent to be tested. Ifthe level of the activity in the presence of the agent differs, by anamount that is statistically significant, from the level of the activityin the absence of the agent, then the agent is an agent that alters theactivity.

This invention further pertains to novel agents identified by theabove-described screening assays. Accordingly, it is within the scope ofthis invention to further use an agent identified as described herein inthe methods of treatment described herein. For example, an agentidentified as described herein can be used to alter Miro1 degradationwith the agent identified as described herein.

For example, in screening assays for biologically active agents, cellsexpressing the mitochondrial transport protein of interest are contactedwith a candidate agent of interest and the effect of the candidate agenton the expression or function of the mitochondrial transport protein isassessed by monitoring one or more mitochondria-associated parameters.In particular the activity of a candidate agent can be assessed bydetermining the level of Miro1 following mitochondria depolarization.

Parameters are quantifiable components of cells, particularly componentsthat can be accurately measured, desirably in a high throughput system.A parameter can be any cell component or cell product including cellsurface determinant, receptor, protein or conformational orposttranslational modification thereof, lipid, carbohydrate, organic orinorganic molecule, nucleic acid, e.g. mRNA, DNA, etc. or a portionderived from such a cell component or combinations thereof. While mostparameters will provide a quantitative readout, in some instances asemi-quantitative or qualitative result will be acceptable. Readouts mayinclude a single determined value, or may include mean, median value orthe variance, etc. Characteristically a range of parameter readoutvalues will be obtained for each parameter from a multiplicity of thesame assays. Variability is expected and a range of values for each ofthe set of test parameters will be obtained using standard statisticalmethods with a common statistical method used to provide single values.Thus, for example, one such method may comprise contacting a cell thatexpresses mitochondrial transport proteins with a candidate agent; andcomparing the mitochondria-associated parameter to themitochondria-associated parameter in a cell that expresses themitochondrial transport proteins but was not contacted with thecandidate agent, wherein a difference in the parameter in the cellcontacted with the candidate agent indicates that the candidate agentwill treat the Parkinson's Disease.

One example of a mitochondria-associated parameter that may bequantified when screening for agents that will treat Parkinson's Diseasewould be the phosphorylation state of a Miro protein (Ser156, Thr298 orThr299 of Miro1 and Miro2), a TRAK protein, or Khc (Ser residues, see,e.g., Lee and Hollenbeck J Biol Chem. 1995 270(10):5600-5), by forexample, immunoprecipitation with mitochondrial transportprotein-specific antibodies followed by Western blotting withphospho-specific and general antibodies where an increase inphosphorylation of Miro proteins and/or a decrease in total Miro levels,or a decrease in phosphorylation of Khc following contact with thecandidate agent indicates that the candidate agent may treat Parkinson'sDisease. Another example of a parameter would be the state ofubiquitination of a Miro protein, a TRAK protein, or Khc, by, forexample, immunoprecipitation with mitochondrial transportprotein-specific antibodies followed by Western blotting withubiquitin-specific antibodies, where an increase in ubiquitinationfollowing contact with the candidate agent indicates that the agent willtreat Parkinson's Disease. Yet another example would be the rate atwhich mitochondria are transported around the cell, measurable by, forexample live cell imaging techniques, where a decrease in the rate oftransport after contacting the cell with candidate agent indicates thatthe agent will treat Parkinson's Disease. Another example would be thelength of the mitochondria in a cell, where a decrease in the length ofthe mitochondria after contacting the cell with candidate agentindicates that the agent will treat Parkinson's Disease. Other outputparameters could include those that are reflective of the ability ofMiro proteins, TRAK proteins, and khc to form a complex (see e.g.,Brickley and Stephenson J. Biol Chem 286(20): 18079-92 (2011)) in thepresence of the MIRO1 reducer, which may be assessed by, for example,co-immunoprecipitation or affinity purification techniques, where adecrease in complex formation after contacting the cell with candidateagent indicates that the agent will treat Parkinson's Disease. In someinstances, one parameter is measured. In some instances, multipleparameters are measured.

All cells comprise mitochondria, and thus, any cell may be used in thesubject screening methods. In some instances, the cell is a cell typethat is typically affected by Parkinson's Disease, e.g. a muscle cell ora neuron, e.g. a motor neuron. In certain instances, the cell comprisesa genetic mutation, i.e. a mutation in a nuclear or mitochondrial gene,which is associated with Parkinson's Disease. In some instances, thecell may be acutely cultured from a subject that has Parkinson'sDisease.

Candidate agents of interest are biologically active agents thatencompass numerous chemical classes, primarily organic molecules, whichmay include organometallic molecules, inorganic molecules, geneticsequences, etc. An important aspect of the invention is to evaluatecandidate drugs, select therapeutic antibodies and protein-basedtherapeutics, with preferred biological response functions. Candidateagents comprise functional groups necessary for structural interactionwith proteins, particularly hydrogen bonding, and typically include atleast an amine, carbonyl, hydroxyl or carboxyl group, frequently atleast two of the functional chemical groups. The candidate agents oftencomprise cyclical carbon or heterocyclic structures and/or aromatic orpolyaromatic structures substituted with one or more of the abovefunctional groups. Candidate agents are also found among biomolecules,including peptides, polynucleotides, saccharides, fatty acids, steroids,purines, pyrimidines, derivatives, structural analogs or combinationsthereof.

Included are pharmacologically active drugs, genetically activemolecules, etc. Compounds of interest include chemotherapeutic agents,anti-inflammatory agents, hormones or hormone antagonists, ion channelmodifiers, and neuroactive agents. Exemplary of pharmaceutical agentssuitable for this invention are those described in, “The PharmacologicalBasis of Therapeutics,” Goodman and Gilman, McGraw-Hill, New York, N.Y.,(1996), Ninth edition, under the sections: Drugs Acting at Synaptic andNeuroeffector Junctional Sites; Drugs Acting on the Central NervousSystem; Autacoids: Drug Therapy of Inflammation; Water, Salts and Ions;Drugs Affecting Renal Function and Electrolyte Metabolism;Cardiovascular Drugs; Drugs Affecting Gastrointestinal Function; DrugsAffecting Uterine Motility; Chemotherapy of Parasitic Infections;Chemotherapy of Microbial Diseases; Chemotherapy of Neoplastic Diseases;Drugs Used for Immunosuppression; Drugs Acting on Blood-Forming organs;Hormones and Hormone Antagonists; Vitamins, Dermatology; and Toxicology,all incorporated herein by reference.

Test compounds include all of the classes of molecules described above,and may further comprise samples of unknown content. Of interest arecomplex mixtures of naturally occurring compounds derived from naturalsources such as plants. While many samples will comprise compounds insolution, solid samples that can be dissolved in a suitable solvent mayalso be assayed. Samples of interest include environmental samples, e.g.ground water, sea water, mining waste, etc.; biological samples, e.g.lysates prepared from crops, tissue samples, etc.; manufacturingsamples, e.g. time course during preparation of pharmaceuticals; as wellas libraries of compounds prepared for analysis; and the like. Samplesof interest include compounds being assessed for potential therapeuticvalue, i.e. drug candidates.

Antibody and small molecules may be screened using a variety of methodsto detect a change in MIRO1 level in vitro and in vivo. e.g., ELISAassays, etc. These methods include, but are not limited to, methods thatmeasure binding affinity to a target, biodistribution of the compoundwithin an animal or cell, or compound mediated cytotoxicity. As a firsttest, the antibody or small molecule may be tested to establishinteraction with a target which signals the level of MIRO1 to change.After selective binding is established, the candidate antibody may betested for appropriate activity in an in vivo model. These and otherscreening methods known in the art provide information on the ability ofa compound to bind to, modulate, or otherwise interact with thespecified target and are a measure of the compound's efficacy. In someembodiments, the method of screening comprises an assay described in anyone of the Examples.

Methods of Diagnosis

In various aspects, a subject is screened to determine if the subject issuffering from or prone to a neurodegenerative disorder such asParkinson's disease. The screening methods comprise behavioral,biophysical, biochemical, and imaging assays and observations as well asquestionnaires to determine if the subject is at risk for or issuffering from the early stages of a neurodegenerative disorder (e.g.,Parkinson's disease). Biophysical and behavioral observations, such asphysical examination of a subject for outward symptoms of disease can beevaluated independently, or combined with questionnaires andbiochemical/imaging assays. Each individual assay can also be utilizedindependently or combined with biophysical evaluations or other teststhat are known in the art and associated with a particularneurodegenerative disorder/disease. Examples of biochemical assaysinclude genetic screens for mutations and/or polymorphisms (e.g., SNPsanalysis, short tandem repeat analysis), biomarker-based assays, proteinexpression assays, immunohistochemistry assays or any combinationsthereof. Material for biochemical assays can be sampled from all bodilyfluids and tissues. Commonly employed bodily fluids include but are notlimited to blood, serum, plasma, saliva, urine, gastric and digestivefluid, tears, stool, semen, vaginal fluid, interstitial fluids derivedfrom tumorous tissue, and cerebrospinal fluid. Methods of obtainingsamples of bodily tissue and fluids include but are not limited tobiopsy, cheek swabbing, nose swabbing, rectal swabbing, skin fatextraction or other collection strategies for obtaining a biological orchemical substance. In some embodiments, the sample is a tissue sample.For example, the tissue sample can be a fibroblast, such as a skinfibroblast.

Control values are measured from corresponding control samples fromcontrol, i.e., non-diseased, subjects. For example, in some embodiments,a MIRO1 level of a skin fibroblast from a subject is compared to acontrol MIRO1 level of a control skin fibroblast from a control subject.

Antibody and small molecule inhibitors may be screened using a varietyof methods to detect target binding in vitro and in vivo. e.g., ELISAassays, etc. These methods include, but are not limited to, methods thatmeasure binding affinity to a target, biodistribution of the compoundwithin an animal or cell, or compound mediated cytotoxicity. As a firsttest, the antibody or small molecule may be tested for binding againstthe target mitochondrial transport protein. After selective binding isestablished, the candidate antibody may be tested for appropriateactivity in an in vivo model. These and other screening methods known inthe art provide information on the ability of a compound to bind to,modulate, or otherwise interact with the specified target and are ameasure of the compound's efficacy.

a subjecta subjecta subjectA method to determine the MIRO1 status of asubject can comprise measuring the Miro1 response to mitochondrialdepolarization using biochemical assays, western blotting or ELISA, todetermine if a subject is deficient in the removal of MIRO1 followingdepolarization, wherein a subject deficient in the removal of MIRO1following depolarization is selected for treatment by administration ofa MIRO1 reducer. Determining the MIRO1 status can comprise detectingMIRO1 level in a subject, and comparing the MIRO1 level to a controlMIRO1 level in a control subject. In some embodiments, detecting MIRO1level comprises detecting MIRO1 in a tissue sample, such as a skinfibroblast, in the subject. In some embodiments, the method comprises anassay described in any one of the Examples.

The method can further comprise treating the subject with the MIRO1reducer. In such instances, the MIRO1 levels can be monitored beforeand/or after treatment with the MIRO1 reducer. In some embodiments, theMIRO1 reducer is administered to the midbrain and/or putamen of thesubject. The treatment can comprise the step of administering to thesubject a therapeutically effective amount of one or more agentsselected from the group consisting of levodopa, a dopamine agonist, aMAO-B inhibitor, amantadine, or an anticholinergic prior to,concurrently with, or after administering the inhibitor of amitochondrial transport protein.

Where the MIRO1 status indicates presence of Parkinson's disease, insome embodiments, the Parkinson's disease is a PTEN-induced putativekinase 1 (PINK-1)-associated form of PD. In some cases, the Parkinson'sdisease is associated with a mutation in any of Parkin, leucine-richrepeat kinase 2, alpha-Synuclein, parkinson protein 7, ATPase type 13A2,phospholipase A2, group VI, DnaJ (Hsp40) homolog, subfamily C, member 6,eukaryotic translation initiation factor 4 gamma, 1, F-box protein 7,GRB10 interacting GYF protein 2, HtrA serine peptidase 2, synaptojanin1, or vacuolar protein sorting 35 homolog. The Parkinson's disease canbe a sporadic form.

The MIRO1 reducer may be administered alone or in combination with anypharmaceutically acceptable carrier or salt known in the art and asdescribed below.

The method of detecting MIRO1 level can be by any method known in theart. In some embodiments, the method of detecting MIRO1 level compriseselectrophoresis, a chromatographic method, an enzyme assay, a bindingassay, or a combination thereof. In some embodiments, the method ofdetecting MIRO1 level comprises ELISA. In some embodiments, the methodcomprises an assay described in any one of the Examples.

As described herein, MIRO1 status correlates with the presence of or anincreased risk of developing certain neurodegenerative disorders. Insome embodiments, an elevated level of MIRO1 is associated with aneurodegenerative disorder such as Parkinson's disease. In someembodiments, the MIRO1 level in a subject is higher by about 5% or more,about 10% or more, or about 20% or more, for example, about 30% or more,about 40% or more, or about 50% or more, about 60% or more, about 70% ormore, or about 80% or more, e.g. about 90% or more, about 95% or more,about 100% or more, about 200% or more, about 300% or more, about 400%or more, about 500% or more, about 600% or more, about 800% or more,about 1000% or more, about 2000% or more, about 3000% or more, relativeto the control MIRO1 level in a control subject. In some embodiments,the MIRO1 level in a subject is higher by about 20% or more relative tothe control MIRO1 level in a control subject. In some embodiments, theMIRO1 level in a subject is higher by about 30% or more relative to thecontrol MIRO1 level in a control subject.

The subject may be suffering from symptoms of a neurodegenerativedisorder. In one aspect, the present invention provides a method fordiagnosing a subject for a neurodegenerative disorder comprising, (a)obtaining a MIRO1 level from a subject; (b) comparing the MIRO1 level toinformation from a control, wherein the information is predetermined toindicate absence of the neurodegenerative disorder. In some embodiments,the neurodegenerative disorder is Parkinson's disease.

Movement disorders. Movement disorders are commonly classified as thosewith decreased or slow movement (hypokinetic disorders) or increasedmovement (hyperkinetic disorders). The classic and most commonhypokinetic disorder is Parkinson disease. Hyperkinetic disordersinclude tremor, myoclonus, dystonia, chorea, and tics.

Atypical parkinsonism refers to a group of neurodegenerative disordersother than Parkinson disease that have some features of Parkinsondisease but have some different clinical features and a differentpathology. As shown herein, the Miro1 status of a subject, assessed bythe methods described herein, provides a distinction between PD andatypical parkonsinism. Atypical parkinsonism encompassesneurodegenerative disorders such as progressive supranuclear palsy,dementia with Lewy bodies, corticobasal ganglionic degeneration, andmultiple system atrophy. Deficits that suggest neurodegenerativedisorders other than Parkinson disease include gaze palsies, signs ofcorticospinal tract dysfunction (eg, hyperreflexia), myoclonus,autonomic dysfunction (if early or severe), cerebellar ataxia, prominentdystonia, ideomotor apraxia (inability to mimic hand motions), earlydementia, early falls, and confinement to a wheelchair.

Parkinson's disease. Parkinson's disease (PD) also known as idiopathicor primary parkinsonism, hypokinetic rigid syndrome/HRS, or paralysisagitans, is a degenerative disorder of the central nervous system. Themotor symptoms of Parkinson's disease result from the death ofdopamine-generating neurons in the substantia nigra, a region of themidbrain, and putamen; the cause of this cell death is unknown. Early inthe course of the disease, the most obvious symptoms aremovement-related; these include shaking, rigidity, resting tremors,bradykinesia, postural stability, slowness of movement and difficultywith walking and gait. Later, thinking and behavioral problems mayarise, with dementia, e.g. cognitive impairment, hallucinations,delusion, behavioral abnormalities, depression, disturbance of sleep andwakefulness, commonly occurring in the advanced stages of the disease,whereas depression is the most common psychiatric symptom. Othersymptoms include sensory (loss of smell), sleep (disturbance of sleepand wakefulness) and emotional problems, constipation, hypotension,urinary frequency, impotence and sweating. Parkinson's disease is morecommon in older people, with most cases occurring after the age of 50.

Parkinson's Disease may be of the familial form or the sporadic form. Bya familial form, it is meant that the disease is inherited, i.e. by thepassage of a heritable gene mutation from parent to child through thegametes. A number of different heritable mutations have been associatedwith PD, including for example, mutations in PTEN-induced putativekinase 1 (PINK-1); Parkin (also known as RBR E3 ubiquitin proteinligase, or PARK2); leucine-rich repeat kinase 2 (LRRK2); alpha-Synuclein(SNCA, PARK4); ubiquitin carboxy-terminal hydrolase L1 (UCHL1);parkinson protein 7 (PARK7, DJ-1); ATPase type 13A2 (ATP13A2);phospholipase A2, group VI (PLA2G6); DnaJ (Hsp40) homolog, subfamily C,member 6 (DNAJC6, PARK19); eukaryotic translation initiation factor 4gamma, 1 (EIF4G1, PARK18); F-box protein 7 (FBXO7); GRB10 interactingGYF protein 2 (GIGYF2); HtrA serine peptidase 2 (HTRA2); synaptojanin 1(SYNJ1); or vacuolar protein sorting 35 homolog (VPS35). By a sporadicform, it is meant that the disease occurs sporadically, i.e. due tosporadic mutation of a gene, e.g. one of the aforementioned genes.

In some cases, the subject is not suffering from symptoms of aneurodegenerative disorder. In one aspect, the present inventionprovides a method for detecting an increased risk for aneurodegenerative disorder in a subject comprising, (a) obtaining aMIRO1 level from a subject; (b) comparing the MIRO1 level to informationfrom a control, wherein the information is predetermined to indicateabsence of the neurodegenerative disorder. In some embodiments, theneurodegenerative disorder is Parkinson's disease.

In some embodiments, a method of diagnosing a neurodegenerative disorderin a subject comprises: providing a sample from a subject suspected ofhaving a neurodegenerative disorder; determining the level of MIRO1 inthe sample; comparing the determined level of the MIRO1 to a controllevel of MIRO1; detecting, in the sample from the subject, an elevatedlevel of the MIRO1 compared to the control level of MIRO1; anddiagnosing the presence of a neurodegenerative disorder in the subjectfrom the elevated level of the MIRO1. After the neurodegenerativedisorder is diagnosed, the subject may be treated with a methoddescribed herein or known in the art. In some embodiments, the methodcomprises an assay described in any one of the Examples.

Methods to Assay Miro1 Status

Assays are provided for determining the Miro1 status of a cell orpopulation of cells. Miro1 is normally removed from damaged, e.g.depolarized, mitochondria to facilitate their clearance via mitophagy,but a defect in this degradation can occur, which defect can be detectedusing biochemical assays. The defect in degradation is stronglyassociated with PD or a predisposition to PD. A high percentage ofParkinson's Disease subjects are deficient in the removal of Miro1following depolarization. However, Miro1 is efficiently degraded upondepolarization in control cells. Detection of this PD-associated defectis useful for diagnosis of PD and prognosis of susceptibility to PD,optionally including the step of treating a subject thus diagnosed;monitoring of clinical response to PD following treatment, in thecontext of a clinical trial, etc.; screening drugs for efficacy inreducing this defect in Miro1 degradation, etc. The methods allowassessment of Parkinson's disease accurately, early and in a clinicallypractical way.

The methods of analysis can be made by examining a cell composition forthe presence of a Miro1 polypeptide following mitochondrial uncoupling,including chemically induced depolarization. These assay methods can beperformed with cell lines, cells obtained from a subject, includingwithout limitation biological samples such as fibroblasts, peripheralblood lymphocytes, and the like. The assay is generally performed onviable, i.e. living cells. Fibroblasts are a convenient source of cellsfrom individuals, and can be easily obtained from subjects by aminimally-invasive, painless procedure. Cultured cells may be derivedfrom patient or control samples; and may be modified to generategenetically-modified cells, in vitro differentiated cells, cells exposedto a candidate therapeutic agent; and the like. In some embodiments theassay is performed on a population of cells from a subject to determinethe Miro1 phenotype of the individual.

In addition, cells that have been genetically altered, e.g. bytransfection or transduction with recombinant genes or by antisensetechnology, to provide a gain or loss of genetic function, may beutilized with the invention. Methods for generating genetically modifiedcells are known in the art, see for example “Current Protocols inMolecular Biology”, Ausubel et al., eds, John Wiley & Sons, New York,N.Y., 2000. The genetic alteration may be a knock-out, usually wherehomologous recombination results in a deletion that knocks outexpression of a targeted gene; or a knock-in, where a genetic sequencenot normally present in the cell is stably introduced.

A variety of methods may be used in the present invention to achieve aknock-out, including site-specific recombination, expression ofanti-sense or dominant negative mutations, and the like. Knockouts havea partial or complete loss of function in one or both alleles of theendogenous gene in the case of gene targeting. Preferably expression ofthe targeted gene product is undetectable or insignificant in the cellsbeing analyzed. This may be achieved by introduction of a disruption ofthe coding sequence, e.g. insertion of one or more stop codons,insertion of a DNA fragment, etc., deletion of coding sequence,substitution of stop codons for coding sequence, etc. In some cases theintroduced sequences are ultimately deleted from the genome, leaving anet change to the native sequence.

A cell sample may comprise, for example, at least about 10² cells, atleast about 10³ cells, at least about 10⁴ cells, at least about 10⁵cells, at least about 10⁶ cells, at least about 10⁷ cells, or more.Higher numbers of cells are optionally assayed in multiple aliquots. Thecells are contacted with an agent that uncouples or depolarizesmitochondria. The concentration of agent and time of exposure issufficient to uncouple or depolarize the mitochondria. For example, CCCPat a concentration of from about 10-100 μM, for example from about 20 to80 μM, from about 30 to 50 μM is sufficient. Dosage of other agents mayprovide for activity comparable to these concentrations of CCCP. Thecells are incubated for a period of time sufficient to depolarizemitochondria and initiate clearance, e.g. at least about 1 hour, atleast about 2 hours, and usually not more than about 24 hours, and maybe from about 1 to 24 hours, from about 2 to 20 hours, from about 3 to18 hours, from about 4 to 14 hours, from about 5 to 10 hours, and may befrom about 4 to 8 hours.

In some embodiments the agent is a mitochondria-specific uncoupler, e.g.a protonophore. Agents suitable for this purpose include FCCP, CCCP,DNP, BAM15, etc., as known in the art and described herein. Followingmitochondrial depolarization, the cells are lysed and assessed forlevels of Miro1, where a deficiency in Miro1 degradation relative to acontrol is indicative of an association with PD. The deficiency may beexpressed, for example, as the ratio in Miro1 levels of a cell lysatesubjected to a mitochondrial uncoupling agent relative to the same cellsin the absence of an uncoupling agent. PD associated cells may have atleast 2-fold the Miro1 present, at least 3-fold, at least 4-fold, ormore relative to a normal cell.

A test sample from a subject is assessed for the presence of analteration in Miro1 degradation in response to mitochondrial uncoupling.The term “alteration” in the polypeptide levels, as used herein, refersto an alteration in levels compared with a control sample. A controlsample is a sample that corresponds to the test sample (e.g., is fromthe same type of cells), and is from a subject who is not affected by asusceptibility to PD. An alteration in the level of the polypeptide inthe test sample, as compared with the control sample, is indicative of asusceptibility to PD. Protein levels can be determined by a variety ofmethods, including enzyme linked immunosorbent assays (ELISAs), Westernblots, immunoprecipitations and immunofluorescence, etc.

For example, in one aspect, an antibody capable of binding to thepolypeptide (e.g., as described above), can be used for capture ordetection. Antibodies can be polyclonal, or more preferably, monoclonal.An intact antibody, or a fragment thereof (e.g., Fab or F(ab′)₂) can beused. The term “labeled”, with regard to the probe or antibody, isintended to encompass direct labeling of the probe or antibody bycoupling (i.e., physically linking) a detectable substance to the probeor antibody, as well as indirect labeling of the probe or antibody byreactivity with another reagent that is directly labeled. Examples ofindirect labeling include detection of a primary antibody using afluorescently labeled secondary antibody, enzyme linked assays,radiolabeled antibodies; and the like as known in the art.

Miro1 degradation may be monitored in a variety of ways. Westernblotting analysis, using an antibody that specifically binds to Miro1can be used to identify the presence in a test sample, e.g. followingcell fractionation, and may be quantitative. In other embodiments, ahigh throughput immunoassay is preferred. Conveniently, the removal ofMiro1 is detected in a subject sample by an immunoassay, such as ELISAor other high throughput affinity assays.

In some embodiments a Miro1 assay as described above is utilized for thediagnosis and clinical monitoring of movement disorders, which diseasesinclude, without limitation, Parkinson's disease. In some embodiments,the methods of the invention are used in determining the efficacy of atherapy for treatment of a movement disorder, e.g. in vitro, such asdrug screening assays and the like; at a subject level; in the analysisof a group of subjects, e.g. in a clinical trial format; etc. Clinicaltrial embodiments may involve the comparison of two or more time pointsfor a subject or group of subjects. The patient status is expected todiffer between the two time points as the result of administration of atherapeutic agent, therapeutic regimen, or challenge with adisease-inducing agent to a subject undergoing treatment. The responseof a subject with a movement disorder to therapy is assessed bydetecting the ability of a cell sample from a subject, including withoutlimitation a fibroblast sample, to degrade Miro1 after mitochondriadamage, e.g. mitochondrial depolarization.

In some embodiments, the method comprises identifying a subject ashaving PD or a predisposition to PD, e.g. by criteria described abovefor Miro1 degradation; administering a dose of a therapeutic agent tothe patient, and quantitating the degradation of Miro1 in response tomitochondrial depolarization in at least one patient sample.

In some embodiments, the methods of the invention are used indetermining the efficacy of a therapy for treatment of a movementdisorder, either at a subject level, or in the analysis of a group ofsubjects, e.g. in a clinical trial format. Such embodiments typicallyinvolve the comparison of two time points for a subject or group ofsubjects. The patient status is expected to differ between the two timepoints as the result of a therapeutic agent, therapeutic regimen, ordisease challenge to a subject undergoing treatment.

For regulatory approval of treatments that offer symptomatic benefit inParkinson's disease subjects, clinical trials have used a double-blind,placebo-controlled, parallel group design with fixed or flexible dosingstrategy. A variety of efficacy outcome measures (one or morecombinations of subscales of the Unified Parkinson's Disease RatingScale [UPDRS]) and need for additional symptomatic therapy such asdopaminergic agonists, levodopa, have been used to assess the effects oftreatment.

Exemplary assays for quantification of MIRO1 include those described inHsieh C-H, Li L, Vanhauwaert R, Nguyen K T, Davis M D, Bu G, et al.Miro1 Marks Parkinson's Disease Subset and Miro1 Reducer Rescues NeuronLoss in Parkinson's Models. Cell Metab. 2019; 1131-1140; and ShaltoukiA, Hsieh C-H, Kim M J, Wang X. Alpha-synuclein delays mitophagy andtargeting Miro rescues neuron loss in Parkinson's models. ActaNeuropathol. 2018; 136: 607-620.

Methods of Treatment

Provided herein is a method of treating a neurodegenerative disordercomprising administering to a subject in need thereof a Miro-reducingagent, wherein the Miro-reducing agent reduces Miro, e.g., Miro1, inParkinson's disease cells with depolarized mitochondria to an amountequivalent to or within 5%, within 10%, within 15%, within 20%, within25%, within 30%, within 35%, within 40%, within 45%, within 50%, within55%, within 60%, within 65% or within 70% of the amount of Miro incontrol healthy cells with depolarized mitochondria not contacted withthe Miro-reducing agent.

Neurodegenerative disorders included within the methods of the presentinvention include, but are not limited to neurological disorders thatshare symptoms similar to those seen in Parkinson's disease relateddisorders. In some cases, the neurological disorders may show symptomssimilar to Parkinson's disease, atypical Parkinson's disease orParkinson's plus disease. Examples include but are not limited toDrug-induced Parkinsonism, Progressive supranuclear Palsy, VascularParkinsonism, Dementia with Lewy Bodies, diffuse Lewy body disease,Corticobasal degeneration, multisystem degeneration (Shy-dragersyndrome), Alzheimer's disease, Pick's disease, and progressivesupranuclear palsy (Steel-Richardson syndrome). Other conditions alsoincluded within the methods of the present invention include age-relateddementia and other dementias and conditions with memory loss includingvascular dementia, diffuse white matter disease (Binswanger's disease),dementia of endocrine or metabolic origin, dementia of head trauma anddiffuse brain damage, dementia pugilistica and frontal lobe dementia. Insome cases, the neurological disorder may not respond well todopaminergic treatments and may be caused as a result of variousvascular, drug-related, infectious, toxic, structural and other knownsecondary causes. Drug-induced Parkinsonism may be caused by agents thatblock post-synaptic dopamine D2 receptors with high affinity, such asanti-psychotic and anti-emetic medications and sodium valproate,anti-depressants, reserpine, tetrabenazine etc.

A variety of subjects are suitable for treatment with an agentidentified by a method of the present disclosure. Suitable subjectsinclude any subject who displays symptoms of Parkinson's disease such asbradykinesia, repetitive movements, tremors, limb rigidity, gait andbalance problems, inability to aim the eyes due to weakness of eyemuscles, weakness, sensory loss, non-motor manifestations such as REMsleep behavior disorder, neuropsychiatric symptoms including mooddisturbances and cognitive changes, anxiety, apathy, changes in thinkingability, level of attention or alertness and visual hallucinations,intellectual and functional deterioration, forgetfulness, personalitychanges, autonomic dysfunction affecting cardiovascular, respiratory,urogenital, gastrointestinal and sudomotor function, difficulties inbreathing and swallowing, inability to sweat, orthostatic hypotension,pain, constipation and loss of olfaction. In some cases, the subjectsmay experience predominant speech or language disorder, predominantfrontal presentation and gait freezing.

In certain embodiments, the subject may not display any overt symptomsof Parkinson's disease. In some cases, the subject in need may showincreased susceptibility to infections, hypothermia, weaker bones, jointstiffness, arthritis, stooped posture, slowed movements, decrease inoverall energy, constipation, urinary incontinence, memory loss, slowerthinking, slower reflexes, difficulty with balance, decrease in visualacuity, diminished peripheral vision, hearing loss, wrinkling skin,greying hair, weight loss, loss of muscle tissue.

In some cases, the subjects may be those who have been diagnosed ashaving Alzheimer's disease; subjects who have suffered one or morestrokes; subjects who have suffered traumatic head injury; individualswho have high serum cholesterol levels; subjects who haveproteinopathies including deposits in brain tissue; subjects who havehad one or more cardiac events; subjects undergoing cardiac surgery; andsubjects with multiple sclerosis.

In some cases, the subject may display symptoms associated withneurological diseases that include motor neuron diseases such asamyotrophic lateral sclerosis, degenerative ataxias, cortical basaldegeneration, ALS-Parkinson's-Dementia complex of Guam, subacutesclerosing panencephalitis, Huntington's disease, Parkinson's disease,synucleinopathies, primary progressive aphasia, striatonigraldegeneration, Machado-Joseph disease/spinocerebellar ataxia type 3 andolivopontocerebellar degenerations, Gilles De La Tourette's disease,bulbar and pseudobulbar palsy, spinal and spinobulbar muscular atrophy(Kennedy's disease), primary lateral sclerosis, familial spasticparaplegia, Werdnig-Hoffmann disease, Kugelberg-Welander disease,Tay-Sach's disease, Sandhoff disease, familial spastic disease,Wohlfart-Kugelberg-Welander disease, spastic paraparesis, progressivemultifocal leukoencephalopathy, and prion diseases (includingCreutzfeldt-Jakob, Gerstmann-Sträussler-Scheinker disease, Kuru andfatal familial insomnia). Also other neurodegenerative disordersresulting from cerebral ischemia or infaction including embolicocclusion and thrombotic occlusion as well as intracranial hemorrhage ofany type (including, but not limited to, epidural, subdural,subarachnoid and intracerebral), and intracranial and intravertebrallesions (including, but not limited to, contusion, penetration, shear,compression and laceration). In some cases, Miro levels uponmitochondrial depolarization in cells from subjects in comparison toMiro levels upon mitochondrial depolarization in cells from controlhealthy subjects may be used as diagnostic assay to identify subjectsthat may benefit from a prophylactic use of the Miro-reducing agent toprevent aberrations in mitochondrial homeostasis. Mitochondrialhomeostasis refers to a balance between the processes of mitochondrialbiogenesis, mitophagy, trafficking, fission, fusion and maintenance ofmitochondrial function and other processes that are at least partlydependent on Miro function.

Mitochondrial depolarization, as used here in this disclosure, refers tothe process by which the potential difference across the mitochondrialmembrane is reduced from its steady state level and the membranepotential of the mitochondria changes in the depolarizing direction fromthe resting potential, from negative to positive. The voltage gradientacross the mitochondrial inner membrane is in part affected by themitochondria permeability transition pore. In some cases, depolarizationbelow a certain ΔΨm may indicate impaired mitochondrial function and caninitiate mitophagy. In some cases, mitochondrial depolarization canprecede the translocation of proteins such as Parkin and Pink1. In somecases, there can be a release of cytochrome accompanying mitochondrialdepolarization. Mitochondrial depolarization can be monitored byrhodamine 123, Mitotracker Red, DiOC₆ or tetramethylrhodamine methylester (TMRE), or methyl ester (TMRM), nonylacridine orange (NAO),Saphranine O, merycyanine 540, JC-1 or JC-9 among others. Mitochondrialdepolarization may be accompanied by up to 5%, up to 7%, up to 10%, upto 15%, up to 18%, up to 20%, up to 25%, up to 30%, up to 35%, up to40%, up to 45%, up to 50%, up to 55%, up to 60%, up to 65%, up to 70%,up to 75%, up to 80%, up to 90%, up to 95% or up to 100% reduction inresting mitochondrial potential, as measured in a quantitative assay.

Mitochondrial depolarization can be induced by depolarization agent.Mitochondrial depolarization agents can be calcium dysregulation, ROSproduction, chemicals such as barbiturates, ginsenoside-Rh2, rotonene,complex-I inhibitors, complex-II inhibitors, complex-III inhibitors,Complex IV inhibitors, mycotoxins such as aurovertins A-E,leucinostatins A and B, venturicidin and ossamycin, efrapeptin,oligomycins A-D, vancomycin, antimycin, naturally occurring flavonoids,propranolol, local anesthetics, herbicide paraquat, pyrethroid, DDT,parathion, diethylstilbestrol, several cationic dyes and organotincompounds, uncouplers such as substituted phenols, carbonyl cyanide4-(trifluoromethoxy) phenylhydrazone (FCCP), carbonyl cyanidemeta-chlorophenylhydrazone (CCCP), trifluoromethylbenzimidazoles,salicylanilides and carbonyl cyanide phenyl hydrazones, endogenous andexogenous free fatty acids (FFA) and fatty-acid like compounds such asperfluorodecanoic acid, sulfuramide and methyl-substitutedhexadecanedioic acid, peptides such as adenine nucleotide translocase,ionophores such as gramicidins (gramicidin A, D and S), nigericin,valinomycin, cationic uncouplers such as cyanine dye tri-S-C4(5),Cu2′-(o-phenanthroline)2 complex, and pentamidine, membrane activepeptides such as alamethicin, Mastoparan, alternative electron acceptorssuch as adriamycin, paraquat, and variously substituted naphthoquinonesand nitrosoamines among others.

In one aspect, the disclosure provides a method of measuring Mirodegradation in cells obtained from subjects having a diagnosis ofsporadic or familial neurodegeneration, under conditions wherein themitochondrial membrane permeability of the cells is altered. In somecases, the cells are fibroblasts derived from tissue obtained fromsubjects diagnosed with sporadic or familial neurodegeneration, themethod comprising obtaining tissue from the patient, preferably skinbiopsy, dissecting the skin biopsy pieces into evenly sized pieces,transferring the dissected skin biopsy pieces into tissue culture platescoated with gelatin, and replacing media with complete DMEM/20% FBSmedia every 2-3 days until fibroblasts are confluent. In some cases, thefibroblasts are obtained from subjects diagnosed with a sporadic orfamilial form of Parkinson's disease. In some cases, theneurodegenerative disorder is Parkinson's disease or Parkinson's-likedisease. In some cases, the subject has the disease and is asymptomatic.In some cases, the subject has a risk factor for the disease and isasymptomatic. The term sporadic Parkinson's disease, as used herein,refers to non-familial forms of Parkinson's disease. In some cases,sporadic PD is caused by environmental factors. In some cases, subjectswith sporadic PD may carry genes associated with PD that have a lowpenetrance or an age of onset late in life which makes any familialoccurrence less obvious.

In some cases, the cells are subject-specific induced pluripotent stemcells (iPSCs) related to Parkinson's disease or Parkinson's-likedisease. In one embodiment the induced pluripotent stem cells arederived from human fibroblasts. In some cases, the subjects can have agenetic form of the disease or a sporadic form of the disease. In arelated embodiment the induced pluripotent stem cells are producedwithout the use of a retrovirus or a lentivirus. In a specificembodiment the induced pluripotent stem cells are produced with a methodcomprising the use of three factors, e.g., OCT4, SOX2, and KLF4. Inanother embodiment, the induced pluripotent stem cells are furtherdifferentiated to adopt a midbrain dopaminergic cell fate. In somecases, the induced pluripotent stem cells are differentiated to adoptthe cell fate in about 20 days.

In some embodiments, the cells are neuronal cells derived from a subjecthaving a diagnosis of Parkinson's disease or Parkinson's-like disease,the method comprising obtaining fibroblasts from the subject,dedifferentiating the fibroblasts into pluripotent stem cells, anddifferentiating the stem cells towards a neuronal cell fate. In oneembodiment, the fibroblasts are dermal fibroblasts. In anotherembodiment the stem cells are differentiated towards a midbraindopaminergic cell fate. In particular embodiments, the dedifferentiationof the fibroblasts induces pluripotency.

In some embodiments, the subject carries a genetic variation or mutationknown to be associated with Parkinson's disease or Parkinson's-likedisease. In other embodiments the genetic variation of interest is acopy number variation of a gene of interest. In specific embodiments,the cell line has three copies of the gene of interest. The geneticvariation or mutation can be a deletion, an insertion, a complexmulti-state variant, a deletion, a substitution, a transition, atransversion, or a duplication, of one or more nucleotides in the geneof interest. In exemplary embodiments, the gene of interest is selectedfrom PARK1 (SNCA or α-synuclein), PARK2 (Parkin), PARKS (UCHL1), PARK6(PINK1), PARK7 (DJ-1), PARK8 (LRRK2), and PARK 11 (GIGFY2). In specificembodiments the gene of interest is PARK1 (SNCA or α-synuclein). Inother specific embodiments, the gene of interest is PARK8 (LRRK2). Inone embodiment , the subject carries a homozygous mutation of LRRK2 andthe mutation comprises a G2019S mutation. In some embodiments, thesubject has Parkinson's disease. In a specific embodiment the subjecthas an idiopathic form of Parkinson's disease.

In some aspects, the methods include providing cells with sequencevariations or multiple copies of the gene of interest, and inducingpluripotency, multipotency or totipotency in the cells to make a cellline with the variation of the gene of interest. The methods may alsoinclude identifying a subject with a variation of the gene of interestand obtaining one or more cells from the subject. The subject-derivedcells may be fibroblast cells, tumor cells, bone marrow cells, stomachcells, blood cells (such as white blood cells, blood progenitor cells),liver cells, etc. or any convenient or relevant source of cells to beobtained from the subject. The methods of generating cell lines with acopy number variation of a gene of interest may also include inducingdifferentiation of the cell line. The cell lines may be differentiatedinto any cell type of interest including endodermal, ectodermal,mesodermal, for example neural, such as neuronal cell lines, epithelialcell lines, cardiac cell lines, etc.

In some aspects, the cell lines and methods of their use include celllines with a genetic copy number variation that is at least oneduplication, for example, two or three copies of a gene. In otheraspects, the copy number variation is one or more deletion, insertion,or complex multi-state variant of the gene of interest.

In some aspects, the cell lines and methods of their use include celllines with a genetic variation that is a mutation of the gene ofinterest. For example, the mutation may be a deletion, substitution,transition, transversion, or duplication of one or more nucleotides inthe gene of interest. In some embodiments, the mutation is a pointmutation.

In one aspect, the disclosure herein provides a method of treating aneurodegenerative disorder comprising administering to a subject in needthereof a Miro-reducing agent, wherein the Miro-reducing agent reducesMiro1 in sporadic Parkinson's disease cells according to an assaycomprising the following steps:

(a) plating fibroblast cells from a sporadic Parkinson's disease subjectinto wells of an array;(b) 24 hours after step (a), a candidate agent is added to a first testwell and the candidate agent is not added to a first control well;(c) 10 hours after step (b), FCCP is added to a first test well andfirst control well;(d) 14 hours after step (c), cells of the first test well and firstcontrol well are fixed with ice-cold 90% methanol; and(e) the cells of the first test well and first control well areimmunostained with anti-Miro1 and 4′,6-diamidino-2-phenylindole,dihydrochloride (DAPI) and imaged with a confocal microscope to measureMiro1 intensity/cell for those images of the first test well and firstcontrol well.

In some cases, the Miro-reducing agent reduces Miro1 in the first testwell by up to three, up to four, up to five, up to six or up to sevenstandard deviations relative to the first control well. In some cases,the Miro-reducing agent reduces Miro1 in the first test well by fromabout 2 to about 7, or about 2 to about 5, or about 2 to 4, or about 2to 6 standard deviations relative to the first control well. In somecases, the candidate agent reduces Miro1 in the second test well by lessthan one or less than a half of a standard deviation relative to thesecond control well, the candidate agent is a Miro-reducing agent. Insome cases, Parkinson's disease cells are obtained from a familialParkinson's disease subject. In some cases, the Parkinson's diseasecells show elevated levels of Miro compared to cells from a healthysubject. In some cases, the Parkinson's disease cells shown comparablelevels of Miro to a healthy subject. In some cases, the mitochondrialdepolarization agent is not FCCP. In some examples, the Miro-reducingagent is a protein or protein fragment, antibody or antibody fragment,peptide, small molecule or aptamer. In some cases, the Miro is detectedby ELISA or Western blotting, or fluorescence live imaging methods.

In one aspect, the disclosure provides a method of treating aneurodegenerative disorder comprising administering to a subject in needthereof a Miro-reducing agent, wherein the Miro-reducing agent reducesMiro1 in sporadic Parkinson's disease cells according to the followingassay:

(a) fibroblast cells from a sporadic Parkinson's disease subject areplated into wells of an array;(b) 24 hours after step (a), a candidate agent is added to a first testwell and the candidate agent is not added to a first control well;(c) 10 hours after step (b), FCCP is added to a first test well andfirst control well;(d) 14 hours after step (c), cells of the first test well and firstcontrol well are fixed with ice-cold 90% methanol; and (e) the cells ofthe first test well and first control well are immunostained withanti-Miro1 and 4′,6-diamidino-2-phenylindole, dihydrochloride (DAPI) andimaged with a confocal microscope to measure Miro1 intensity/cell forthose images of the first test well and first control well. In oneaspect, the disclosure provides method for treatment of aneurodegenerative disorders comprising administering to a subject inneed thereof a Miro-reducing agent, wherein Miro-reducing agent reducesMiro1 in sporadic Parkinson's disease cells with depolarizedmitochondria by two, three, four, five or six standard deviations ormore relative to sporadic Parkinson's disease cells with depolarizedmitochondria not treated with the Miro-reducing agent.

In some cases, the Miro-reducing agent identified in the above assaydoes not reduce Miro1 in sporadic Parkinson's disease cells according tothe following assay:

(a1) fibroblast cells from a sporadic Parkinson's disease subject areplated into wells of an array;(b1) 24 hours after step (a1), the candidate agent is added to a secondtest well and is not added to a second control well;(c1) 14 hours after step (b1), cells of the second test well and secondcontrol well are fixed with ice-cold 90% methanol; and(d1) the cells of the second test well and second control well areimmunostained with anti-Miro1 and 4′,6-diamidino-2-phenylindole,dihydrochloride (DAPI) and imaged with a confocal microscope to measureMiro1 intensity/cell for those images of the second test well and secondcontrol well. In one aspect, the disclosure provides method fortreatment of a neurodegenerative disorder comprising administering to asubject in need thereof a Miro-reducing agent, wherein Miro-reducingagent reduces Miro1 in sporadic Parkinson's disease cells withdepolarized mitochondria by two, three, four, five or six standarddeviations or more relative to sporadic Parkinson's disease cells withdepolarized mitochondria not treated with the Miro-reducing agent, andreduces Miro in sporadic Parkinson's disease cells by less than onestandard deviation compared to sporadic Parkinson's disease cells nottreated with the Miro-reducing agent.

In one aspect, the invention described herein provides a method oftreating a neurodegenerative disorder comprising administering to asubject in need thereof a Miro-reducing agent, wherein the Miro-reducingagent reduces Miro1 in Parkinson's disease cells with depolarizedmitochondria. In some cases, the Miro-reducing agent reduces Miro1 inParkinson's disease cells with depolarized mitochondria, to an amountequivalent to or within 15%, 17.5%, 20%, 22.5%, 25%, 27.5%, 30%, 32.5%,35%, 37.5%, 40%, 42.5%, 45%, 47.5%, 50%, 52.5%, 55%, 57.5%, 60%, 62.%,65%, 67.5%, 70%, 72.5%, 75% of the amount of Miro in control healthycells with depolarized mitochondria not contacted with the Miro-reducingagent. In some cases, the Miro-reducing agent reduces Miro inParkinson's disease cells to an amount between 15%-25%, 15%-30%,20%-30%, 20%-40%, 25%-40%, 25%-35%, 25%-45%, 30%-45%, 30%-50%, 35%-55%,35%-60%, 35%-50%, 35%-55%, 40%-50%, 40%-55%, 40%-60%, 45%-65%, 45%-70%,45%-75% of the amount of Miro in control healthy cells with depolarizedmitochondria not contacted with the Miro-reducing agent. In some cases,the healthy cells with depolarized mitochondria are healthy cellscontacted with a mitochondrial depolarizing agent.

In one aspect, the disclosure provides a method of treating aneurodegenerative disorder comprising administering to a subject in needthereof a Miro-reducing agent, wherein the Miro-reducing agent reducesMiro1 in Parkinson's disease cells with depolarized mitochondriacompared to reduction of Miro1 in control depolarized sporadicParkinson's disease cells with depolarized mitochondria not contactedwith the Miro-reducing agent. In some cases, the Miro-reducing agentreduces Miro1 in Parkinson's disease cells with depolarized mitochondriaby three standard deviations or more as compared to reduction of Miro1in control depolarized sporadic Parkinson's disease cells withdepolarized mitochondria not contacted with the Miro-reducing agent. Insome cases, the Miro-reducing agent reduces Miro1 in Parkinson's diseasecells with depolarized mitochondria by two standard deviations or moreas compared to reduction of Miro1 in control depolarized sporadicParkinson's disease cells with depolarized mitochondria not contactedwith the Miro-reducing agent. In some cases, the Miro-reducing agentreduces Miro1 in Parkinson's disease cells with depolarized mitochondriaby four standard deviations or more as compared to reduction of Miro1 incontrol depolarized sporadic Parkinson's disease cells with depolarizedmitochondria not contacted with the Miro-reducing agent. In some cases,the Miro-reducing agent reduces Miro1 in Parkinson's disease cells withdepolarized mitochondria by five standard deviations or more as comparedto reduction of Miro1 in control depolarized sporadic Parkinson'sdisease cells with depolarized mitochondria not contacted with theMiro-reducing agent.

In one aspect, the disclosure provides for a method of treating aneurodegenerative disorder comprising administering to a subject in needthereof a Miro-reducing agent, wherein (a) the Miro-reducing agentreduces Miro1 in Parkinson's disease cells with depolarized mitochondriaby two standard deviations or more as compared to reduction of Miro1 incontrol Parkinson's disease cells with depolarized mitochondria notcontacted with the Miro-reducing agent; and (b) the Miro 1 reducingagent reduces Miro1 in Parkinson's disease cells with non-depolarizedmitochondria by one standard deviation or less as compared to reductionof Miro1 in control Parkinson's disease cells with non-depolarizedmitochondria not contacted with the Miro-reducing agent. In some cases,the Miro-reducing agent reduces Miro1 in Parkinson's disease cells withdepolarized mitochondria by three standard deviations or more ascompared to reduction of Miro1 in control depolarized sporadicParkinson's disease cells with depolarized mitochondria not contactedwith the Miro-reducing agent. In some cases, the Miro-reducing agentreduces Miro1 in Parkinson's disease cells with depolarized mitochondriaby four standard deviations or more as compared to reduction of Miro1 incontrol depolarized sporadic Parkinson's disease cells with depolarizedmitochondria not contacted with the Miro-reducing agent. In some cases,the Miro-reducing agent reduces Miro1 in Parkinson's disease cells withdepolarized mitochondria by five standard deviations or more as comparedto reduction of Miro1 in control depolarized sporadic Parkinson'sdisease cells with depolarized mitochondria not contacted with theMiro-reducing agent. In some cases, the Parkinson's disease cells withdepolarized mitochondria are Parkinson's disease cells contacted with amitochondrial depolarizing agent. In some cases, the Parkinson's diseasecells with depolarized mitochondria are Parkinson's disease cells notcontacted with a mitochondrial depolarizing agent. In some cases, theParkinson's disease cells are sporadic Parkinson's disease cells. Insome cases, the Parkinson's disease cells are familial Parkinson'sdisease cells. In some cases, the Parkinson's disease cells arefibroblasts. In some cases, the Parkinson's disease cells show elevatedlevels of Miro compared to cells from a healthy subject. In some cases,the Parkinson's disease cells shown comparable levels of Miro to ahealthy subject. In some cases, the mitochondrial depolarization agentis not FCCP. In some examples, the Miro-reducing agent is a protein orprotein fragment, antibody or antibody fragment, peptide, small moleculeor aptamer. In some cases, the Miro is detected by ELISA or Westernblotting, or fluorescence live imaging methods.

In some cases, the subject has a higher Miro level, e.g., a higher Miro1level, than the corresponding Miro level in a control subject. Forexample, the subject can have about 20% or more, about 30% or more,about 40% or more, about 50% or more, about 60% or more, about 70% ormore, about 80% or more, about 90% or more, about 100% or more, about200% or more, about 300% or more, about 400% or more, about 500% ormore, about 1000% or more, higher level of Miro1 than a control subject.In some cases, the subject can have a higher level of Miro1 by about 20%or more or about 30% or more compared with the Miro1 level in a controlsubject.

In some cases, the candidate agent is a peptide, antibody, protein,protein fragment, aptamer or small molecule. In one embodiment, thescreening assays of the invention are high throughput or ultra highthroughput. For example, the screening assays of the invention a may becarried out in a multi-well format, for example, a 96-well, 384-wellformat, or 1,536-well format, and are suitable for automation. Inparticular, each well of a microtiter plate can be used to run aseparate assay against a selected test agent. In some cases, theconcentration or incubation time effects of a single test are to beobserved, every 5-10 wells can test a single test agent. It is possibleto assay many plates per day; assay screens for up to about 6,000,20,000, 50,000, or more than 100,000 different compounds using thismethod.

Candidate agents can be proteins or protein fragments, antibody orantibody fragment, small molecules or aptamers. The candidate agent canbe in the form of a library of candidate agents, such as a combinatorialor randomized library that provides a sufficient range of diversity.Candidate agents are optionally linked to a fusion partner, e.g.,targeting compounds, label or detectable moiety, rescue compounds,dimerization compounds, stabilizing compounds, addressable compounds,and other functional moieties. In some cases, the candidate agent isattached to the surface of an array. In some cases, the candidate agentsare delivered in a liquid sample to the cells expressing Miro.

In some cases, the Parkinson's cells and the healthy cells express Miroprotein linked to a label or reporter moiety. A “label” or a “detectablemoiety” is a composition detectable by spectroscopic, photochemical,biochemical, immunochemical, chemical, or other physical means. Forexample, useful labels include ³²P, fluorescent dyes, electron-densereagents, enzymes (e.g., as commonly used in an ELISA), biotin,digoxigenin, or haptens and proteins which can be made detectable, e.g.,by incorporating a radiolabel into the peptide or used to detectantibodies specifically reactive with the peptide. In some cases, levelsof Miro may be detected by the use of fluorescence, luminescence,chemiluminescence, absorbance, and other optical methods. In some cases,the difference in Miro levels between the Parkinson's disease and thehealthy cells is measured by computer algorithms.

In some cases, the cells may be engineered to express Miro proteintagged to enzymatic reporters that can generate a fluorescent signal orare capable of binding small molecules that can be tagged with afluorescent moiety. In some examples, the small molecule may be taggedwith a fluorescent reagent such as fluorescein, rhodamine, Texas Red,BODIPY and other commercially available molecules (such as thoseavailable from Molecular Probes/Invitrogen and other suppliers) avariety of fluorescent readouts can be generated. In other examples,ligands and other probes can be tagged directly with fluorescein oranother fluorophore for detection of binding to cellular proteins; orcan be tagged with enzymes such as alkaline phosphatase or horseradishperoxidase to enable indirect detection and localization of signal. Inother examples, Miro may be tagged with enzymes that can be used togenerate a fluorescent signal in live cells by using a specific,cell-permeable substrate that either becomes fluorescent or shifts itsfluorescent spectrum upon enzymatic cleavage, resulting in shifts influorophore absorption or emission wavelengths. In other examples, Miromay be tagged with enzymes that can be used to generate a fluorescentsignal in live cells by cleavage of a covalent assembly ofemission-absorption-matched fluorophore pairs that in thecovalently-assembled form sustain resonance energy transfer between thetwo fluorophores that is lost when the two are separated.

Luminescent, fluorescent or bioluminescent signals are easily detectedand quantified with any one of a variety of automated and/orhigh-throughput instrumentation systems including fluorescencemulti-well plate readers, fluorescence activated cell sorters (FACS) andautomated cell-based imaging systems that provide spatial resolution ofthe signal. A variety of instrumentation systems have been developed toautomate HCS including the automated fluorescence imaging and automatedmicroscopy systems developed by Cellomics, Amersham, TTP, Q3DM, Evotec,Universal Imaging and Zeiss. Fluorescence recovery after photobleaching(FRAP) and time lapse fluorescence microscopy have also been used tostudy protein mobility in living cells.

In one aspect, the invention also provides methods for identifying adiagnostic cellular phenotype comprising comparing a set of cells from asubject to cells from a subject free of Parkinson's disease wherein, thecellular phenotype detected is the degradation of Miro uponmitochondrial depolarization. In some cases, the comparison of Mirolevels is performed on a computer. The cells may be fibroblasts. Thecells may be induced pluripotent stem cells, or cells differentiatedfrom induced stem cells to neural stem cells, or neurons. In oneembodiment, the detected response is a change in mitochondrial function,mitochondrial fission, fusion, morphology, mitophagy, mitochondrialtransport, intracellular calcium levels or other cellular features thatare dependent on Miro function.

In one aspect, the invention also involves methods for determining therisk of Parkinson's disease in a subject comprising comparing at leastone phenotype determined in a first set of cells derived from thesubject to the at least one phenotype determined in a second set ofcells derived from subjects free of Parkinson's disease and to the atleast one phenotype determined in a third set of cells derived fromsubjects suffering from Parkinson's disease; and indicating that thesubject is at high risk for Parkinson's disease if the at least onephenotype determined in the first set of cells is more similar to the atleast one phenotype determined in the third set of cells than the atleast one phenotype determined in the second set of cells, wherein thefirst, second, and third sets of cells were induced pluripotent stemcells, or were cells differentiated from induced pluripotent stem cells,and wherein the phenotype is the degradation of Miro upon mitochondrialdepolarization. In one embodiment, the detected response is a change inmitochondrial function, mitochondrial fission, fusion, morphology,mitophagy, mitochondrial transport or other cellular features that aredependent on Miro function. In some cases, the comparison is performedon a computer. In some cases, the Parkinson's disease cells showelevated levels of Miro compared to cells from a healthy subject. Insome cases, the Parkinson's disease cells shown comparable levels ofMiro to a healthy subject. In some cases, the mitochondrialdepolarization agent is not FCCP. In some examples, the Miro-reducingagent is a protein or protein fragment, antibody or antibody fragment,peptide, small molecule or aptamer. In some cases, the Miro is detectedby ELISA or Western blotting, or fluorescence live imaging methods.

In one aspect, the disclosure provides for a method for selecting asubject for treatment with a therapeutic agent for a neurodegenerativedisorder, comprising:

(a) collecting cells from the subject and evaluating a first controlportion of the cells for the pre-depolarization Miro level in the cells;(b) contacting a second test portion of the cells with a depolarizingagent; and(c) evaluating the post-depolarization Miro level in the second testportion of cells contacted with the depolarizing agent and comparing theMiro level to the pre-depolarization Miro1 level in the first controlportion of cells.

In some cases, the subject is treated with a therapeutic agent forneurodegenerative disorder when the post-depolarization Miro1 level inthe second test portion of cells is reduced by 5% to 70%, 10% to 70%,20% to 80%, 25% to 90%, 5% to 50%, or 10% to 50%, relative to thepre-depolarization Miro level in the first control portion of cells. Incertain embodiments, the depolarizing agent reduces post-depolarizationMiro level in the second test portion of cells by 5% up to 40%, up to35%, up to 30%, up to 25%, up to 20%, up to 15%, relative to thepre-depolarization Miro level in the first control portion of cells.

In some cases, the neurodegenerative disorder is Parkinson's disease. Insome cases, the cells are fibroblasts. In some cases, the cells areinduced pluripotent stem cells or cells differentiated from inducedpluripotent stem cells. In some cases, the cells are neuronsdifferentiated from induced pluripotent stem cells. In some cases, theneurons are dopaminergic neurons. In some cases, the depolarizationagent is FCCP.

The present invention involves methods for identifying an agent thatcorrects a phenotype associated with a Parkinson's disease or apredisposition for Parkinson's disease comprising contacting a firstpopulation of cells with a candidate agent, contacting a secondpopulation of cells with a control agent, assaying the two populationsand identifying candidate agents as correcting the phenotype if thefirst population is closer to a normal phenotype following treatmentthan the second population, where the phenotype is cellular degradationof Miro and lowering of Miro levels following mitochondrialdepolarization. In some cases, the candidate agent is a Miro-reducingagent. In some cases, the cells in both populations have been treatedwith a mitochondrial depolarization agent. In some cases, the cells inboth populations comprise at least one endogenous allele associated witha neurodegenerative disorder or associated with a predisposition forneurodegeneration. In some cases, the Parkinson's disease cells showelevated levels of Miro compared to cells from a healthy subject. Insome cases, the Parkinson's disease cells show comparable levels of Miroto a healthy subject. In some cases, the mitochondrial depolarizationagent is not FCCP. In some examples, the Miro-reducing agent is aprotein or protein fragment, antibody or antibody fragment, peptide,small molecule or aptamer. In some cases, the Miro is detected by ELISAor Western blotting, or fluorescence live imaging methods.

In one aspect, the disclosure provides a method of screening candidateagents to identify a Miro-reducing agent, comprising the steps of:

(a) obtaining cells deficient in Miro1 clearance;(b) contacting a first test portion of the cells with a candidate agentand not contacting a second control portion of the cells with thecandidate agent;(c) contacting the first test portion and second control portion ofcells with of step (b) with a depolarizing agent;(d) evaluating the Miro1 levels in the first test portion relative tothe second control portion.

In some cases, the candidate agent is identified as a Miro-reducingagent if the candidate agent decreases Miro levels in the first testportion by two or more, three or more, four or more, five or more, sixor more, or seven or more standard deviations relative to the Mirolevels in the second control portion. In some cases, the first andsecond test portion comprise cells obtained from a subject identifiedwith Parkinson's disease or at risk for Parkinson's disease. In somecases, the cells are fibroblasts. In some cases, the cells are inducedpluripotent stem cells or cells differentiated from pluripotent stemcells. In some cases, the cells are neurons differentiated frompluripotent stem cells. In one aspect, the disclosure provides a methodof treating or preventing Parkinsonism disorders by administering to asubject an Miro-reducing agent identified by the screening methodsdescribed herein an effective amount to treat or prevent Parkinson'sdisease.

In one aspect, the invention provides methods for reducing the risk ofdrug toxicity in a human subject of Parkinsonism, comprising contactingone or more cells generated from the subject, with a dose of apharmacological agent, assaying the contacted one or more differentiatedcells for toxicity, and prescribing or administering the pharmacologicalagent to the subject if, and only if, the assay is negative for toxicityin the contacted cells. The cells may be fibroblasts from the subjects,induced pluripotent cells generated from the subject, or cellsdifferentiated from the induced pluripotent stem cell line such asneurons.

In one aspect, the disclosure provides for a method to screen cell lineswith variations of a gene of interest for a candidate agent to treat aneurodegenerative disorder. The methods include contacting cells derivedfrom a subject deficient in or suspected of being deficient in theremoval of MIRO1 with a candidate agent, observing a change or lack ofchange in the cells, and correlating a change or lack of change with theability of the agent to treat the disease. Such changes may be observed,for example, by staining for intracellular levels of Miro upondepolarization. The methods may further comprise comparing the cell lineor its progeny with a cell line or its progeny without the variation inthe gene of interest, that is, a normal cell line, or a cell linecorrelated to the same disorder of interest, but without the variationin the gene of interest present in the first cell line. The cells may befibroblasts from the subjects, induced pluripotent cells generated fromthe subject, or cells differentiated from the induced pluripotent stemcell line such as neurons.

Methods of studying the mechanism of a neurodegenerative disorder suchas Parkinson's disease are also provided herein. The methods includeidentifying a molecular determinant of the disorder or disease bycontacting a cell line or its progeny made by the methods describedherein with an agent or condition which affects a cellular pathway ofinterest and observing a change or lack of change in the cells. In somecases, the cellular pathway of interest is Miro degradation uponmitochondrial depolarization. In one embodiment, the cellular pathway isa change in mitochondrial function, mitochondrial fission, fusion,morphology, mitophagy, mitochondrial transport or other cellularfeatures that are dependent on Miro function.

In some cases, the Miro-reducing agent binds to the EF hand domain ofMiro. In some cases, the Miro-reducing agent increases intracellularcalcium in the cell. In some cases, the Miro-reducing agent binds to theGTPase domain of Miro. In some cases, the Miro-reducing agent binds tothe nucleotide binding domain of Miro. In some cases, the Miro-reducingagent binds to the microtubule binding region of Miro. In some cases,Miro-reducing agent binds to the Pink1 phosphorylation site of Miro.

In some cases, the Miro-reducing agent is a calcium channel blocker.Calcium channels are protein molecules containing pores extendingthrough the membranes of cells or cellular organelles, which reversiblyopen and close, thus regulating the passage of Ca++ ions into and out ofthe cell or organelle. Known calcium channels include L-type, N-type,and R-type calcium channels.

In some cases, the calcium channel is an L-type channel. L-type channelsare characterized by (1) “high threshold” for activation, i.e., a strongdepolarization of the cell membrane in which they are located isrequired to open such channels; (2) large “single channel conductance”,i.e., each channel, when opened, can allow the passage of Ca+2 ions at arelatively high rate; (3) greater permeability to Ba+2 than Ca+2; and,of particular note, (4) sensitivity to high potency block by thedihydropyridine class of calcium channel antagonists such as nimodipineand nifedipine (characteristically the IC50 values for L-channel blockby these drugs are below 1 μM). In most cases, the calcium “actionpotentials” mediated by L-type channels under normal physiologicalcircumstances is of relatively long duration, typically no less than 100msec. In some cases, the Miro-reducing agent is a L-type channelblocker.

In some cases, the calcium channel is an N-type channel. N-type channelsare high threshold channels which are most appropriately described asdihydropyridine-insensitive but blocked by interaction with the conesnail toxin omega-conotoxin. Qualitatively, as a class, N-type channelsinactivate somewhat more rapidly than L-type channels. Because there isoverlap between L- and N-type channel classes in this regard,differences in inactivation kinetics do not constitute a defin-ingcharacteristic. In some cases, the Miro-reducing agent is a N-typechannel.

In some cases, the calcium channel is an R-type channel. R-typechannels, may be characterized as high-threshold calcium channels whichare relatively resistant to block by dihydropyridines andomega-conotoxin. Such channels are found in a wide variety of neurons,and are particularly abundant in cerebellar Purkinje cells. R-typechannels may play a role in synaptic transmission and other processesthat depend on calcium entry but are not sensitive to these blockers. Insome cases, the Miro-reducing agent is a R-type channel.

In some cases, the Miro-reducing agent may be a calcium channelantagonist capable of reversibly blocking calcium channels. In somecases, the Miro-reducing agent may function by nonpermanently (i.e.,noncovalently) binding to the protein molecules which constitute suchchannels. In some cases, the Miro-reducing agent may be a calciumchannel antagonist which blocks calcium channels to a greater degreethan it blocks neurotransmitter-activated channels, voltage-sensitivesodium channels and potassium channels: i.e., its IC50 for calciumchannels is lower than that for such neurotransmitter-activated, sodiumand potassium channels. In some cases, the Miro-reducing agent can crossthe blood-brain barrier of a subject. In some cases, the Miro-reducingagent may be used to treat neurodegenerative disorders associated withexcessive calcium influx into neuronal cells, including Parkinson'sdisease.

In some examples, the effect of a Miro-reducing agent on intracellularcalcium levels may be monitored by organic synthetic fluorescent dyessuch as Fura-2, Fluo-3, Fluo-4, Indo-1, Calcium green-1, Oregon greenBAPTA, Rhod-2. In some examples, the effect of a Miro-reducing agent onintracellular calcium levels may be monitored by Aequorin-basedluminescence calcium indicators such as Aequorin. In other examples, theeffect of a Miro-reducing agent on intracellular calcium levels may bemonitored by fluorescent protein based calcium indicators, such asDsRed/inverse pericam, YC 2.1, G-Camp, YC 3.1, G-Camp2, Synapcam, YC2.1,YC 2.12, Camgaroo, G-CaMP2, G-CaMP2, YC 3.12, Cer TN-L15, GCamp3 etc.

Pharmaceutical Compositions

In some embodiments, a pharmaceutical composition comprising aneffective dose of Miro1 reducer is provided, which dose may besufficient to achieve a therapeutic level of Miro1 of at least 1 μM, atleast 5 μM, at least 10 μM, at least 20 μM, up to about 1 mM, up toabout 500 μM, up to about 250 μM, up to about 100 μM, up to about 75 μM,up to about 50 μM. A unit dose may be, for example, 1 μg/kg, 10 μg/kg,100 μg/kg, 500 μg/kg, 1 mg/ kg, 5 mg/kg, 10 mg/kg, 50 mg/kg, 100 mg/kg,or more.

The term “pharmaceutically acceptable” means approved by a regulatoryagency of the Federal or a state government or listed in the U.S.Pharmacopeia or other generally recognized foreign pharmacopeia for usein animals, and more particularly in humans. The term “carrier” or“vehicle” refers to a diluent, adjuvant, excipient, or vehicle withwhich the Miro1 reducer is administered. Such pharmaceutical carrierscan be, for example, lipids, e.g. liposomes, e.g. liposome dendrimers;sterile liquids, such as saline solutions in water and oils, includingthose of petroleum, animal, vegetable or synthetic origin, such aspeanut oil, soybean oil, mineral oil, sesame oil and the like. A salinesolution is a preferred carrier when the pharmaceutical composition isadministered intravenously. Saline solutions and aqueous dextrose andglycerol solutions can also be employed as liquid carriers, particularlyfor injectable solutions. Suitable pharmaceutical excipients includestarch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk,silica gel, sodium stearate, glycerol monostearate, talc, sodiumchloride, dried skim milk, glycerol, propylene glycol, water, ethanoland the like. The composition, if desired, can also contain minoramounts of wetting or emulsifying agents, or pH buffering agents. Thesecompositions can take the form of solutions, suspensions, emulsion,tablets, pills, capsules, powders, sustained-release formulations andthe like. The composition can be formulated as a suppository, withtraditional binders and carriers such as triglycerides. The reducer canbe formulated as neutral or salt forms. Pharmaceutically acceptablesalts include those formed with free amino groups such as those derivedfrom hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., andthose formed with free carboxyl groups such as those derived fromsodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine,triethylamine, 2-ethylamino ethanol, histidine, procaine, etc. Examplesof suitable pharmaceutical carriers are described in “Remington'sPharmaceutical Sciences” by E. W. Martin, hereby incorporated byreference herein in its entirety. Such compositions will contain atherapeutically effective amount of the Miro1 reducer, preferably inpurified form, together with a suitable amount of carrier so as toprovide the form for proper administration to the patient. Theformulation should suit the mode of administration.

The pharmaceutical composition can also include any of a variety ofstabilizing agents, such as an antioxidant for example. When thepharmaceutical composition includes a polypeptide, the polypeptide canbe complexed with various well-known compounds that enhance the in vivostability of the polypeptide, or otherwise enhance its pharmacologicalproperties (e.g., increase the half-life of the polypeptide, reduce itstoxicity, enhance solubility or uptake). Examples of such modificationsor complexing agents include sulfate, gluconate, citrate and phosphate.The polypeptides of a composition can also be complexed with moleculesthat enhance their in vivo attributes. Such molecules include, forexample, carbohydrates, polyamines, amino acids, other peptides, ions(e.g., sodium, potassium, calcium, magnesium, manganese), and lipids.

Further guidance regarding formulations that are suitable for varioustypes of administration can be found in Remington's PharmaceuticalSciences, Mace Publishing Company, Philadelphia, Pa., 17th ed. (1985).For a brief review of methods for drug delivery, see, Langer, Science249:1527-1533 (1990).

The components used to formulate the pharmaceutical compositions arepreferably of high purity and are substantially free of potentiallyharmful contaminants (e.g., at least National Food (NF) grade, generallyat least analytical grade, and more typically at least pharmaceuticalgrade). Moreover, compositions intended for in vivo use are usuallysterile. To the extent that a given compound must be synthesized priorto use, the resulting product is typically substantially free of anypotentially toxic agents, particularly any endotoxins, which may bepresent during the synthesis or purification process.

The subject pharmaceutical composition is typically sterile. Sterilityis readily accomplished by filtration through sterile filtrationmembranes (e.g., 0.2 μm membranes). Therapeutic compositions generallyare placed into a container having a sterile access port, for example,an intravenous solution bag or vial having a stopper pierceable by ahypodermic injection needle. The pharmaceutical composition may bestored in unit or multi-dose containers, for example, sealed ampules orvials, as an aqueous solution or as a lyophilized formulation forreconstitution. As an example of a lyophilized formulation, 10-mL vialsare filled with 5 ml of sterile-filtered 1% (w/v) aqueous solution ofcompound, and the resulting mixture is lyophilized. The pharmaceuticalcomposition comprising the lyophilized Miro1 reducer is prepared byreconstituting the lyophilized compound, for example, by usingbacteriostatic Water-for-Injection.

The pharmaceutical composition can be formulated for intravenous, oral,via implant, transmucosal, transdermal, intramuscular, intrathecal, orsubcutaneous administration. In some embodiments, the pharmaceuticalcomposition is formulated for intravenous administration. In otherembodiments, the pharmaceutical composition is formulated forsubcutaneous administration. The following delivery systems, whichemploy a number of routinely used pharmaceutical carriers, are onlyrepresentative of the many embodiments envisioned for administering theinstant compositions.

Components of the pharmaceutical composition can be supplied eitherseparately or mixed together in unit dosage form, for example, as a drylyophilized powder or water free concentrate. Where the composition isto be administered by infusion, it can be dispensed with an infusionbottle containing sterile pharmaceutical grade water or saline. Wherethe composition is administered by injection, an ample of sterile waterfor injection or saline can be provided so that the ingredients may bemixed prior to administration.

In some embodiments, the pharmaceutical composition is supplied as a drysterilized lyophilized powder that is capable of being reconstituted tothe appropriate concentration for administration to a subject. In someembodiments, the pharmaceutical composition is supplied as a water freeconcentrate. In some embodiments, the pharmaceutical composition issupplied as a dry sterile lyophilized powder at a unit dosage of atleast 0.5 mg, at least 1 mg, at least 2 mg, at least 3 mg, at least 5mg, at least 1 0 mg, at least 15 mg, at least 25 mg, at least 30 mg, atleast 35 mg, at least 45 mg, at least 50 mg, at least 60 mg, or at least75 mg.

Methods of Administration

In the subject methods, the active agent(s) may be administered to thesubject using any convenient means capable of resulting in the desiredreduction in impairment of mitochondrial integrity and/or function,reduction in any associated neurological disorder, etc.

Thus, the agent can be incorporated into a variety of formulations fortherapeutic administration. More particularly, the agents of the presentinvention can be formulated into pharmaceutical compositions bycombination with appropriate, pharmaceutically acceptable carriers ordiluents, and may be formulated into preparations in solid, semi-solid,liquid or gaseous forms, such as tablets, capsules, powders, granules,ointments, solutions, suppositories, injections, inhalants and aerosols.

In pharmaceutical dosage forms, the agents may be administered in theform of their pharmaceutically acceptable salts, or they may also beused alone or in appropriate association, as well as in combination,with other pharmaceutically active compounds. The following methods andexcipients are merely exemplary and are in no way limiting.

For oral preparations, the agents can be used alone or in combinationwith appropriate additives to make tablets, powders, granules orcapsules, for example, with conventional additives, such as lactose,mannitol, corn starch or potato starch; with binders, such ascrystalline cellulose, cellulose derivatives, acacia, corn starch orgelatins; with disintegrators, such as corn starch, potato starch orsodium carboxymethylcellulose; with lubricants, such as talc ormagnesium stearate; and if de-sired, with diluents, buffering agents,moistening agents, preservatives and flavoring agents.

The agents can be formulated into preparations for injection bydissolving, suspending or emulsifying them in an aqueous or nonaqueoussolvent, such as vegetable or other similar oils, synthetic aliphaticacid glycerides, esters of higher aliphatic acids or propylene glycol;and if desired, with conventional additives such as solubilizers,isotonic agents, suspending agents, emulsifying agents, stabilizers andpreservatives.

The agents can be utilized in aerosol formulation to be administered viainhalation. The com-pounds of the present invention can be formulatedinto pressurized acceptable propellants such as dichlorodifluoromethane,propane, nitrogen and the like.

Furthermore, the agents can be made into suppositories by mixing with avariety of bases such as emulsifying bases or water-soluble bases. Thecompounds of the present invention can be administered rectally via asuppository. The suppository can include vehicles such as cocoa butter,carbowaxes and polyethylene glycols, which melt at body temperature, yetare solidified at room temperature.

Unit dosage forms for oral or rectal administration such as syrups,elixirs, and suspensions may be provided wherein each dosage unit, forexample, teaspoonful, tablespoonful, tablet or suppository, contains apredetermined amount of the composition containing one or moreinhibitors. Similarly, unit dosage forms for injection or intravenousadministration may comprise the inhibitor(s) in a composition as asolution in sterile water, normal saline or another pharmaceuticallyacceptable carrier.

The term “unit dosage form,” as used herein, refers to physicallydiscrete units suitable as unitary dosages for human and animalsubjects, each unit containing a predetermined quantity of compounds ofthe present invention calculated in an amount sufficient to produce thedesired effect in association with a pharmaceutically acceptablediluent, carrier or vehicle. The specifications for the novel unitdosage forms of the present invention depend on the particular compoundemployed and the effect to be achieved, and the pharmacodynamicsassociated with each compound in the subject.

Other modes of administration will also find use with the subjectinvention. For instance, an agent of the invention can be formulated insuppositories and, in some cases, aerosol and intranasal compositions.For suppositories, the vehicle composition will include traditionalbinders and carriers such as, polyalkylene glycols, or triglycerides.Such suppositories may be formed from mixtures containing the activeingredient in the range of about 0.5% to about 10% (w/w), prefer-ablyabout 1% to about 2%.

Intranasal formulations will usually include vehicles that neither causeirritation to the nasal mucosa nor significantly disturb ciliaryfunction. Diluents such as water, aqueous saline or other knownsubstances can be employed with the subject invention. The nasalformulations may also contain preservatives such as, but not limited to,chlorobutanol and benzalkonium chloride. A surfactant may be present toenhance absorption of the subject proteins by the nasal mucosa.

An agent of the invention can be administered as injectables. Typically,injectable compositions are prepared as liquid solutions or suspensions;solid forms suitable for solution in, or suspension in, liquid vehiclesprior to injection may also be prepared. The preparation may also beemulsified or the active ingredient encapsulated in liposome vehicles.

Suitable excipient vehicles are, for example, water, saline, dextrose,glycerol, ethanol, or the like, and combinations thereof. In addition,if desired, the vehicle may contain minor amounts of auxiliarysubstances such as wetting or emulsifying agents or pH buffering agents.Actual methods of preparing such dosage forms are known, or will beapparent, to those skilled in the art. See, e.g., Remington'sPharmaceutical Sciences, Mack Publishing Company, Easton, Pa., 17thedition, 1985; Remington: The Science and Practice of Pharmacy, A. R.Gennaro, (2000) Lippincott, Williams & Wilkins. The composition orformulation to be administered will, in any event, contain a quantity ofthe agent adequate to achieve the desired state in the subject beingtreated.

The pharmaceutically acceptable excipients, such as vehicles, adjuvants,carriers or diluents, are readily available to the public. Moreover,pharmaceutically acceptable auxiliary substances, such as pH adjustingand buffering agents, tonicity adjusting agents, stabilizers, wettingagents and the like, are readily available to the public.

Routes of Administration

Conventional and pharmaceutically acceptable routes of administrationinclude intranasal, intra-muscular, intratracheal, intratumoral,subcutaneous, intradermal, topical application, intravenous, rectal,nasal, oral and other parenteral routes of administration. Routes ofadministration may be combined, if desired, or adjusted depending uponthe agent and/or the desired effect. The com-position can beadministered in a single dose or in multiple doses.

The agent can be administered to a subject using any availableconventional methods and routes suitable for delivery of conventionaldrugs, including systemic or localized routes. In general, routes ofadministration contemplated by the invention include, but are notnecessarily limited to, enteral, parenteral, or inhalational routes.

Parenteral routes of administration other than inhalation administrationinclude, but are not necessarily limited to, topical, transdermal,subcutaneous, intramuscular, intraorbital, intracapsular, intraspinal,intrasternal, and intravenous routes, i.e., any route of administrationother than through the alimentary canal. Parenteral administration canbe carried to effect systemic or local delivery of the agent. Wheresystemic delivery is desired, administration typically involves invasiveor systemically absorbed topical or mucosal administration ofpharmaceutical preparations.

The agent can also be delivered to the subject by enteraladministration. Enteral routes of administration include, but are notnecessarily limited to, oral and rectal (e.g., using a suppository)delivery.

Methods of administration of the agent through the skin or mucosainclude, but are not necessarily limited to, topical application of asuitable pharmaceutical preparation, transdermal transmission, injectionand epidermal administration. For transdermal transmission, absorptionpromoters or iontophoresis are suitable methods. Iontophoretictransmission may be accomplished using commercially available “patches”which deliver their product continuously via electric pulses throughunbroken skin for periods of several days or more.

Kits

Also provided are reagents, devices and kits thereof for practicing oneor more of the above-described methods. The subject reagents, devicesand kits thereof may vary greatly. Reagents and devices of interestinclude those mentioned above with respect to the methods of treatingParkinson's Disease in a subject.

Kits with unit doses of the active agent, e.g. in oral or injectabledoses, are provided. In such kits, in addition to the containerscontaining the unit doses will be an informational package insertdescribing the use and attendant benefits of the drugs in treatingpathological condition of interest. Preferred compounds and unit dosesare those described herein above.

In one embodiment, the kit includes a MIRO1 reducer and apharmaceutically acceptable carrier. In particular embodiments, theMIRO1 reducer and the pharmaceutically acceptable carrier are packagedseparately. For example, the MIRO1 reducer may be included in the kit ina dry form, packaged in a container or vial, separate from the carrier.In other embodiments, the MIRO1 reducer is formulated in apharmaceutically acceptable carrier.

In certain embodiments, the kit further includes at least one additionaltherapeutic agent. In particular embodiments, the additional therapeuticagent is selected from the group consisting of levodopa, a dopamineagonist, a MAO-B inhibitor, amantadine, an anticholinergic, a PUM1antagonist, an SR protein antagonist, a Parkin agonist, a PINK1 agonist,an 4E-BP1 agonist, a Drp1 agonist, an Atg1 agonist, a TauS2A agonist, aRbf1 agonist, a Dp antagonist, an E2f1 antagonist, a Polo-like kinase 2antagonist and a Notch agonist.

In addition to the above components, the subject kits will furtherinclude instructions for practicing the subject methods of diagnosis ortreatment (e.g., instructions regarding route of administration, dose,dosage regimen, site of administration, and the like). Theseinstructions may be present in the subject kits in a variety of forms,one or more of which may be present in the kit. One form in which theseinstructions may be present is as printed information on a suitablemedium or substrate, e.g., a piece or pieces of paper on which theinformation is printed, in the packaging of the kit, in a packageinsert, etc. Yet another means would be a computer readable medium,e.g., diskette, CD, etc., on which the information has been recorded.Yet another means that may be present is a website address which may beused via the internet to access the information at a removed site. Anyconvenient means may be present in the kits.

Crossing the Blood-Brain Barrier

The blood-brain barrier limits the uptake of many therapeutic agentsinto the brain and spinal cord from the general circulation. Moleculeswhich cross the blood-brain barrier use two main mechanisms: freediffusion; and facilitated transport. Because of the presence of theblood-brain barrier, attaining beneficial concentrations of a giventherapeutic agent in the central nervous system (CNS) may require theuse of drug delivery strategies. Delivery of therapeutic agents to theCNS can be achieved by several methods.

One method relies on neurosurgical techniques. In the case of gravelyill subjects such as accident victims or those suffering from variousforms of dementia, surgical intervention is warranted despite itsattendant risks. For instance, therapeutic agents can be delivered bydirect physical introduction into the CNS, such as intraventricular orintrathecal injection of drugs. Intraventricular injection may befacilitated by an intraventricular catheter, for example, attached to areservoir, such as an Ommaya reservoir. Methods of introduction may alsobe provided by rechargeable or biodegradable devices. Another approachis the disruption of the blood-brain barrier by substances whichincrease the permeability of the blood-brain barrier. Examples includeintra-arterial infusion of poorly diffusible agents such as mannitol,pharmaceuticals which increase cerebrovascular permeability such asetoposide, or vasoactive agents such as leukotrienes. Neuwelt andRappoport (1984) Fed. Proc. 43:214-219; Baba et al. (1991) J. Cereb.Blood Flow Metab. 11:638-643; and Gennuso et al. (1993) Cancer Invest.11:638-643.

Further, it may be desirable to administer the pharmaceutical agentslocally to the area in need of treatment; this may be achieved by, forexample, local infusion during surgery, by injection, by means of acatheter, or by means of an implant, said implant being of a porous,non-porous, or gelatinous material, including membranes, such assilastic membranes, or fibers.

Therapeutic compounds can also be delivered by using pharmacologicaltechniques including chemical modification or screening for an analogwhich will cross the blood-brain barrier. The compound may be modifiedto increase the hydrophobicity of the molecule, decrease net charge ormolecular weight of the molecule, or modify the molecule, so that itwill resemble one normally transported across the blood-brain barrier.Levin (1980) J. Med. Chem. 23:682-684; Pardridge (1991) in: Peptide DrugDelivery to the Brain; and Kostis et al. (1994) J. Clin. Pharmacol.34:989-996.

Encapsulation of the drug in a hydrophobic environment such as liposomesis also effective in delivering drugs to the CNS. For example WO91/04014 describes a liposomal delivery system in which the drug isencapsulated within liposomes to which molecules have been added thatare normally transported across the blood-brain barrier.

Another method of formulating the drug to pass through the blood-brainbarrier is to encapsulate the drug in a cyclodextrin. Any suitablecyclodextrin which passes through the blood-brain barrier may beemployed, including, but not limited to, J-cyclodextrin, K-cyclodextrinand derivatives thereof. See generally, U.S. Pat. Nos. 5,017,566,5,002,935 and 4,983,586. Such compositions may also include a glycerolderivative as described by U.S. Pat. No. 5,153,179.

Delivery may also be obtained by conjugation of a therapeutic agent to atransportable agent to yield a new chimeric transportable therapeuticagent. For example, vasoactive intestinal peptide analog (VIPa) exertedits vasoactive effects only after conjugation to a monoclonal antibody(Mab) to the specific carrier molecule transferrin receptor, whichfacilitated the uptake of the VI-Pa-Mab conjugate through theblood-brain barrier. Pardridge (1991); and Bickel et al. (1993) Proc.Natl. Acad Sci. USA 90:2618-2622. Several other specific transportsystems have been identified, these include, but are not limited to,those for transferring insulin, or insulin-like growth factors I and II.Other suitable, non-specific carriers include, but are not limited to,pyridinium, fatty acids, inositol, cholesterol, and glucose derivatives.Certain prodrugs have been described whereby, upon entering the centralnervous system, the drug is cleaved from the carrier to release theactive drug. U.S. Pat. No. 5,017,566

EXAMPLES Example 1 Assays to Identify Miro-Reducing Agents Cell Culture

Human dermal primary fibroblasts isolated from a PD patient (ND33424)and a healthy control (ND36091) were cultured in cell media DMEM(ThermoFisher, 11995-065) supplemented with 10% fetal bovine serum(Gemini Bio-Products, 900-108, heat-inactivated), 1×Anti Anti(ThermoFisher, 15240096), and 1×GlutaMax (ThermoFisher, 35050061)maintained in a 37° C., 5% CO₂ incubator with humidified atmosphere.

Western Blot

Fibroblasts were cultured in high-glucose DMEM (SH30243.01, Invitrogen)supplemented with 10% heat-inactivated fetal bovine serum (F0926,Sigma-Aldrich, and 900-108, Gemini Bio Products) and maintained in a 37°C., 5% CO₂ incubator with humidified atmosphere. The media wererefreshed every 3-4 days and split every 7-8 days. CCCP (C2759,Sigma-Aldrich) was prepared at 40 mM in DMSO fresh every time andapplied at 40 μM in fresh culture medium (1:1000 dilution). IP wasperformed as described in (Hsieh et al., 2016).

For transfection in fibroblasts, medium was replaced with Opti-MEM(Gibco) prior to transfection. 0.5 μg of DNA or 2 μl of Lipofectamine2000 was diluted in Opti-MEM at room temperature (22° C.) to a finalvolume of 50 μl in two separate tubes, and then contents of the twotubes were gently mixed, incubated for 25 min at room temperature, andsubsequently added onto fibroblasts. After transfection for 6 hrs,Opti-MEM containing DNA-Lipofectamine complexes was replaced withregular culture medium. After transfection for 18 hrs, fibroblasts werelive imaged, or treated with Miro1 reducer and/or CCCP.

Mitochondria were isolated from cultured human fibroblasts as describedpreviously with minor modifications. Briefly, CCCP in DMSO or the samevolume of DMSO treated fibroblasts were lifted by a cell scraper, andmechanically homogenized with a Dounce homogenizer in 750 μl isolationbuffer (200 mM sucrose, 10 mM TRIS/MOPS, pH 7.4). After centrifugationat 500 g for 10 min, crude supernatant was spun at 10,000 g for 10 minto pellet intact mitochondria. Mitochondrial pellet was washed twicewith isolation buffer. After this step, supernatant was referred to“cytosolic fraction (Cyto)”, and pellet was resuspended in 50 μl lysisbuffer (50 mM Tris pH 8.0, 150 mM NaCl, and 1% Triton X-100—T8787,Sigma-Aldrich) with 0.25 mM phenylmethanesulfonylfluoride (P7626,Sigma-Aldrich) and protease inhibitors (Roche) referred to“mitochondrial fraction (Mito)”.

Samples were mixed 1:1 with 2×laemmli buffer (4% SDS, 20% Glycerol, 120mM Tris-HCl, 0.02% bromophenol blue, 700 mM 2-mercaptoethanol) andboiled for 5 min prior to being loaded (Mito:Cyto=25:1) into anSDS-PAGE. 10% polyacrylamide gels (acrylamide:bis-acrylamide=29:1) andTris-Glycine-SDS buffer (24.8 mM Tris, 192 mM glycine, 0.1% SDS) wereused for electrophoresis.

After electrophoresis, nitrocellulose membranes (1620115, Bio-Rad) wereused in wet transfer with Tris-Glycine buffer (24.8 mM Tris, 192 mMglycine) at 360 mA for 2 hrs on ice. Transferred membranes were firstblocked overnight in phosphate-buffered saline (PBS) containing 5%fat-free milk and 0.1% tween-20 at 4° C., and then incubated with thefollowing primary antibodies: mouse anti-Miro1 (WH0055288M1,Sigma-Aldrich) at 1:1,000, rabbit anti-Miro1 (HPA010687, Sigma-Aldrich)at 1:1,000, rabbit anti-Miro2 (HPA012624, Sigma-Aldrich) at 1:800,rabbit anti-VDAC (4661S, Cell Signaling Technology) at 1:1,000, mouseanti-Mitofusin2 (H00009927-M01, Abnova) at 1:1,000, mouse anti-Parkin(sc32282, Santa Cruz Biotechnology) at 1:500, rabbit anti-LRRK2(NB300-268, Novus Biologicals) at 1:500, rabbit anti-OPA1 (ab42364,Abcam) at 1:750, mouse anti-β-actin (A00702, Genscript) at 1:1,000,mouse anti-ubiquitin (A-104, Boston Biochem) at 1:500 or rabbitanti-GAPDH (5174S, Cell Signaling Technology) at 1:3,000, at 4° C.overnight in blocking buffer. HRP-conjugated goat anti-mouse or rabbitIgG (Jackson ImmunoResearch Laboratories) were used at 1:5-10,000. WestDura ECL Reagents (34075, GE Healthcare) were used for ECLimmunoblotting. Membranes were exposed to UltraCruz autoradiographyfilms (Santa Cruz Biotechnology) and developed on a Konica MinoltaSRX-101A developer. For fluorescent Western, blots were probed withCy5-conjugated goat anti-mouse IgG (PA45009, GE Healthcare) at 1:5,000,and scanned at 635nm with a Molecular Dynamics Storm 860 Imager(Amersham BioSciences, Piscataway, N.J.) in a linear range forfluorescent detection. Representative raw blots are in supplementarytables. Experiments were repeated for more than 3 times.

Statistics of Fibroblast Western Blotting Data. All experiments wereperformed in a blinded format, and the identities of the lines wereun-blinded either by us (NINDS lines) or by the PPMI researchers (PPMIlines). Films were scanned or digital blots were exported as 16-bit tiffformat. The intensities of protein bands were measured by ImageJ (ver.1.48V, NIH). The intensity of each band in the mitochondrial fractionwas normalized to that of the mitochondrial loading control VDAC fromthe same blot, and expressed as a fraction of the mean of Healthy-1 withDMSO treatment; this control was included in every independentexperiment. Values of Mean±S.E.M of Miro1 were reported in Table S1A.Values of Mean of Miro1, Mitofusin2, LRRK2, and Parkin were importedinto heat maps in FIG. 1D. The band intensities of VDAC were notsignificantly different among all fibroblast lines and conditions(p=0.8490, One-Way ANOVA Post-Hoc Tukey Test with adjustment). n=3-35independent experiments. Mann-Whitney U Test was performed for comparingnormalized Miro1 band intensities within the same subject (DMSO v.s.CCCP), and the P values were reported in Table S1A. The numbers ofsubjects with a P value >0.05 and <0.05 were counted respectively andused in Fisher Exact Test in Table A. Linear Regression Analysis wasused to determine the correlation with the ratio of Miro1 intensity(mean intensity at CCCP/DMSO). One-Way ANOVA Post-Hoc Tukey Test withadjustment was performed for band intensities at baseline (P>0.3509 forall markers in “Cyto”+“Mito”). Statistical analyses were two-sided andperformed using the Prism software (ver. 5.01, GraphPad).

ELISA

All experiments were performed as blinded tests. 40 μM CCCP in DMSO orthe same volume of DMSO alone was applied to fibroblasts for 6 hrs, andthen cells were lysed in lysis buffer (100 mM Tris, 150 mM NaCl, 1 mMEGTA, 1 mM EDTA, 1% Triton X-100, 0.5% Sodium deoxycholate) withprotease inhibitor cocktail (539134, Calbiochem). Cell debris wasremoved by centrifugation at 17,000 g for 10 min at 4° C. Microplates(MaxiSorp, NUNC) were coated with mouse anti-Miro1 (clone 4H4,WH0055288M1, Sigma-Aldrich) at 1:1,000, chicken anti-β-actin (LS-C82919,LifeSpan BioSciences) at 1:750, mouse anti-β-actin (A00702, Genscript)at 1:1000, or mouse anti-ATP5β (ab14730, Abcam) at 1:1000 in 0.1 Msodium carbonate-bicarbonate buffer (3:7, pH=9.6) overnight at roomtemperature with cover to avoid evaporation. After plates were washed inwash buffer (0.05% Tween 20 in PBS, pH 7.3), nonspecific binding siteswere blocked in PBS with 2% BSA (BP-1600-100, Fisher scientific) for 1hr. Next, 50 μl of cell lysate prepared from above, purified full-lengthMiro1 protein (0-900 ng/ml, ab163047, Abcam), or serial dilutions ofcell lysates of fibroblasts (Healthy-1) or HEK cells (1/16×-2×) wereadded and incubated at room temperature for 2 hrs. After washes, plateswere incubated with biotinylated rabbit anti-Miro1 (ARP44818_P050, AvivaSystems Biology) at 1:1000, or biotinylated rabbit anti-β-actin (#5057S,Cell Signaling Technology) at 1:500, in 100 μl diluent (1% BSA in PBS,pH=7.3) for 2 hrs. Next, plates were washed and incubated withhorseradish peroxidase-conjugated streptavidin (21130, ThermoScientific) at 1:2000 in 100 μl diluent for 20 min. Plates were washedagain, and 100 μl of the tetramethylbenzidine liquid substrate (SB01,Life Technologies) was added and incubated for another 20 min. Thecolorimetric reactions were stopped by 50 μl 1 M H₂SO4 and absorbancewas read at 450 nm by a microplate reader (FlexStation 3, Moleculardevices). An experiment for generating the standard curve was includedin each plate and representative standard plots were shown in figures.Each data point was from 4 independent experiments with 2 technicalrepeats each time. Mann-Whitney U test was performed for comparing Miro1signals within the same subject (DMSO v.s. CCCP). Basal Miro1 signalswere not significantly different among all lines (P>0.1177, One-WayANOVA Post-Hoc Tukey Test with adjustment). The distribution of datapoints was expressed as box-whisker plots (Extreme, Quartile, Median).

GTPase

IPed Miro1 was eluted from protein-A beads by incubation with 60 μl 0.2M glycine (pH 2.5) for 10 min, followed by centrifugation at 3000 g for2 min. Supernatants (eluates) were collected and neutralized by addingan equal volume of Tris pH 8.0. Eluates were processed for colorimetricanalysis of GTPase activity at room temperature by using a GTPaseActivity Kit according to the manufacturer's instructions (602-0120,Novus Biologicals). 100 μl eluates were mixed with 100 μlsubstrate/buffer mix (20μl 0.5 M Tris Buffer, 5 μl 0.1 M MgCl₂, 10 μl 10mM GTP, and 65 μl ddH₂O). 200 μl of inorganic phosphate group (Pi)standard (0-50 μM) was prepared in water. Next, 50 μl PiColorLock™ mixwas added into either Pi standards or samples. After 2 min, stabilizerwas added and mixed thoroughly. 30 min later, absorbance was read at 650nm by a microplate reader (FlexStation 3, Molecular devices). The assaywas validated using purified Miro1 protein (ab163047, Abcam). Theconcentrations of purified Miro1 protein ranging from 0-900 ng/ml showeda linear dependency on the absorbances at 650 nm which reflect releasedPi (R²=0.9711, P=0.0021). Omitting GTP or Miro1 eliminated the signals(Pi<3 μM). Experiments were repeated twice.

qPCR

Total RNA was extracted from at least 10⁶ cells per sample using TRIzol®(GIBCO) according to the manufacturer's instructions. Concentrations oftotal RNA were measured using a Nano Drop. 1 μg of total RNA was thensubjected to DNA digestion using DNase I (Ambion), immediately followedby reverse transcription using the iScript Reverse TranscriptionSupermix (1708841, BIO-RAD). qPCR was performed using the StepOnePlus™instrument (Thermo Fisher Scientific) and SYBR® Green Supermix(172-5270, BIO-RAD) by following the manufacturer's instructions. HumanGAPDH was amplified as internal standards. Expression levels wereanalyzed by the StepOne™ Software (Version 2.2.2). The relativeexpression level of Miro1 was divided by that of GAPDH from the sameexperiment. Each sample was analyzed in duplicate from 4 independentbiological repeats. The following primers were used:

GAPDH forward: (SEQ ID NO: 1) 5′-ACCACAGTCCATGCCATCAC-3′ GAPDH reverse:(SEQ ID NO: 2) 5′-TCCACCACCCTGTTGCTGT-3′ Miro1 forward: (SEQ ID NO: 3)5′-GGGAGGAACCTCTTCTGGA-3′ Miro1 reverse: (SEQ ID NO: 4)5′-ATGAAGAAAGACGTGCGGAT-3′.

Screening Assay Protocol

Fibroblast cells were plated onto clear-bottomed/black-walled 384-wellplates (EK-30091, E&K Scientific, Santa Clara, Calif.) at 2000cells/well with Matrix Wellmate dispenser (Thermo Scientific, Sunnyvale,Calif.) and plates were incubated at 37° C., 5% CO₂ for 24 hrs. Next,chemical library compounds at defined concentrations, were added usingfully automated liquid handling system Caliper Life Sciences Staccatosystem with a Twister II robot and a Sciclone ALH3000 (Caliper LifeSciences, Alameda, Calif. USA) integrated with a V&P Scientific pin tooland plates were incubated at 37° C./5% CO₂ for 10 hrs. Then, 20 μM ofFCCP (Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone,Sigma-Aldrich, C2920) in cell media was added to defined wells andplates were incubated at 37° C., 5% CO₂ for another 14 hr. Cells werefixed with ice-cold 90% methanol (Fisher, 482332) for 20 min at −20° C.,incubated with blocking buffer (10% Normal Goat Serum (NGS)(ThermoFisher, 50062Z), 0.5% BSA (ThermoFisher, BP1600), 0.2% TritonX-100 (ThermoFisher, T8787) at room temperature (RT) for 15 min, andincubated with anti-Miro1 solution (Sigma-Aldrich, HPA0101687) at 1:100in blocking buffer O/N at 4° C. Samples were washed with lx PBS(ThermoFisher, 10010-049) using a Plate Washer multivalve (Bio-Tek,ELx405UV), incubated with goat anti-Rabbit IgG (H+L) Cross-Adsorbed,Alexa Fluor 488 (ThermoFisher, A11008) at 1:500 in blocking buffer at25° C. for 2 hrs and washed again with lx PBS, finally 1.0 μg/mL DAPI(ThermoFisher, D1306) in 1×PBS was added and plates were sealed usingPlateLoc (Velocity11, 01867.001). All liquids were dispensed using theMultidrop 384 (Titertek, 5840200) unless otherwise stated. Fluorescentsignals in plates were automatically imaged with ImageXpress Micro(Molecular Devices, IXMicro) and data were analyzed with MetaXpressAnalysis (Molecular Devices). The percent of Miro1 reduction wascalculated as % Positive Cells for Miro1/Median=[(Positive Cells MiroIntegrated Intensity/All Nuclear Miro Integrated Intensity)−Plate Medianof this same calculation (Median % Positive Miro/Nuclear Miro in PlateData)]/[Plate Median of this same calculation (Median % PositiveMiro/Nuclear Miro in Plate Data)]. Positive cells are cells having Miro1immunostaining. A zero value indicates no difference from the Median(Inactive). Negative value indicates a reduction in Miro. Positive valueindicates an increase in Miro. The screening and data analysis werecarried out together with Stanford University High-Throughput BioscienceCenters (HTBC).

Hit Confirmation—Immunocytochemistry, Confocal Microscopy, ImageAnalysis

Fibroblast cells were plated in 6-well plates (VWR, 10861-554) oncoverslips (Fisher scientific, 22-293232) at 400.000 cells/well andplates were incubated at 37° C., 5% CO₂ for 24 hrs. Next, newly orderedchemical compounds dissolved in DMSO were added at definedconcentrations and plates were incubated at 37° C./5% CO₂ for 10 hrs.Then, FCCP (final concentration of 20 μM, Carbonyl cyanide4-(trifluoromethoxy)phenylhydrazone, Sigma-Aldrich, C2920) in cell mediawas added to defined wells and plates were incubated at 37° C., 5% CO₂for another 14 hrs. Cells were fixed with ice-cold 90% methanol (Fisher,482332) for 20 min at −20° C., incubated with blocking buffer (10%Normal Goat Serum (NGS) (ThermoFisher, 50062Z), 0.5% BSA (ThermoFisher,BP1600), 0.2% Triton X-100 (ThermoFisher, T8787)) at 25° C. for 15 minand incubated with anti-Miro1 (Sigma-Aldrich, HPA0101687) at 1:100 inblocking buffer O/N at 4° C. Samples were washed three times with 1×PBS(ThermoFisher, 10010-049), incubated with goat anti-Rabbit IgG (H+L)Cross-Adsorbed, Alexa Fluor 488 (ThermoFisher, A11008) at 1:500 inblocking buffer at 25° C. for 2 hrs and washed again three times with lxPBS, and finally samples were mounted with ProLong™ Glass AntifadeMountant with NucBlue™ Stain (hard-setting, Thermofisher, P36983) onglass slides and allowed to cure O/N. Samples were imaged at 25° C. witha 20×/N.A.0.60 oil Plan-Apochromat objective on a Leica SPE laserscanning confocal microscope (JH Technologies), with identical imagingparameters among different genotypes. Images were processed with ImageJ(Ver. 1.48, NIH) using the Intensity Ratio Nuclei Cytoplasm Tool(http://dev.mri.cnrs.fr/projects/imagej-macros/wiki/Intensity_Ratio_Nuclei_Cytoplasm_Tool),and average cytoplasmic intensity (the average intensity in thecytoplasm area) was plotted.

Drugs from the NIH Clinical collection library were screened at adefined 10 μM concentration with 4 biological repeats for each drug (377compounds). 11 out of the 377 unique compounds were found to reduceMiro1 protein levels by three standard deviations, following FCCPtreatment, in all biological repeats. These compounds are ranked in theorder of the degree of Miro1 reduction (Table 1A). 12 small moleculesthat reduced Miro1 protein levels in 3 out of 4 biological repeats wereranked in order of degree of Miro1 reduction. The BioMol FDA library wasscreened at different 5-fold doses (1.25-2.5-5-10-20 μM) and induplicate, which identified 3 hits that partially but significantlyreduced Miro1 protein levels following FCCP treatment in adose-dependent manner. These hits were ranked in order of the degree ofMiro1 reduction, with the six compounds found to reduce Miro1 proteinlevels by 20% or more shown (Table 1A). Moreover, an additional 12 smallmolecules reduced Miro1 protein levels in 3 out of 4 biological repeats(Table 1B). We next screened the Biomol FDA library with 5-fold doses(1.25-2.5-5-10-20 μM) and in duplicate. From 175 unique compounds, weidentified 3 hits that partially but significantly reduced Miro1 proteinlevels following FCCP treatment in a dose-dependent manner. We rankedthese 3 hits in the order of the degree of Miro1 reduction (Table 1A).Taken together, we identified 14 actives (2.5% primary hit rate) and 12potential actives.

To reduce experimental bias such as the freshness the compounds,experimental reagents, and imaging devices, the 14 hits identified inthe high-throughput screening were verified individually . The 14compounds were tested at their highest screening concentrations with atleast 4 biological replicates, using the same sporadic PD line. Miro1levels were evaluated without mitochondrial depolarization (DMSO) butwith the compound treatment, which reveals the drug effect on Miro1 onpolarized healthy mitochondria. Samples were imaged with identicalsettings under the confocal microscope, and negative controls withoutprimary antibodies yielded no signals under the imaging settings. 7 outof the initial 14 hit compounds were found to consistently reduce Miro1protein following FCCP treatment (Table 1A). One compound, Fenbufen,appeared toxic (Table 1A). Of the 7 confirmed hits, compounds Tranilastand Benidipine HCl had no impact on basal Miro1 levels, whereascompounds Physostigmine, Pravastatin Sodium, Lofepramine, Temozolomideand Escitalopram Oxalate already reduced Miro1 protein at baseline.

Example 2 Miro1 Marks Parkinson's Disease Subset

The identification of molecular targets and pharmacodynamic markers forParkinson's disease (PD) will empower more effective clinical managementand experimental therapies. Miro1 is localized on the mitochondrialsurface and mediates mitochondrial motility. Miro1 is removed fromdepolarized mitochondria to facilitate their clearance via mitophagy.Here we explore the clinical utility of Miro1 for detecting PD and forgauging potential treatments. We measure the Miro1 response tomitochondrial depolarization using biochemical assays in skinfibroblasts from a broad spectrum of PD subjects, and discover that morethan 94% of the subjects' fibroblast cell lines fail to remove Miro1following depolarization.

Miro1 Is Resistant to Removal from Depolarized Mitochondria in SkinFibroblasts from a Large Population of PD Subjects. Skin fibroblasts canbe easily obtained from subjects by a minimally-invasive, painlessprocedure. We aimed to determine the frequency of the Miro1 phenotype inskin fibroblasts from a large cohort of both sporadic and familial PDsubjects. We fractioned mitochondria after CCCP treatment thatdepolarizes the mitochondrial membrane potential (ΔΨm). In wild-typecontrols at 6 hrs following treatment, both Miro1 and Mitofusin2 wereremoved from damaged mitochondria as detected by Western blotting (FIG.1A, 1B).

Table S1A. Related to FIG. 1. Demographic Information and Miro1 Values.Demographic information and clinical scores are from the onlinedatabases of the consortia. Not all information is available for allsubjects. Particular analyses use data from the subjects with theavailable information. P values are from comparing the normalized Miro1intensities within the same subject (CCCP v.s. DMSO).

The OMM protein Mitofusin2 is a target of the PINK1-Parkin pathway, butnot of LRRK2, for depolarization-triggered degradation. We includedMitofusin2 in our readout to compare its phenotypic frequency in PD withMiro1's. We screened a total of 71 PD and 3 at-risk fibroblast linescomprising the entire PD fibroblast collection at the National Instituteof Neurological Disorders and Stroke (NINDS) human and cell repositoryand the first released PD-control cohort from the Parkinson'sProgression Markers Initiative (PPMI). All subjects were diagnosed withPD and without the presence of signs for other neurological disorders.At-risk subjects are younger asymptomatic family members of the probandsand harbor the same genetic mutations (in LRRK2 or SNCA). Twenty-eightsubjects have a positive family history. We included 22 controls thatconsisted of 12 age/gender/race-matched healthy subjects recruited fromthe same cohorts and 10 subjects with other neurological disordersincluding Huntington's (HD) or Alzheimer's disease (AD) (Table A). Weconducted our assay in a blinded manner.

TABLE A Summary of the Miro1 Phenotype in All Subjects Used in ThisStudy. No. (Miro1 DMSO No. (Miro1 DMSO P (Fisher Exact) No. confirmedNo. confirmed Disease v.s. CCCP P < 0.05) v.s. CCCP P > 0.05) comparedto PD by ELISA by Western PD 5 78 (94%) 24 73 PD Risk 0  5 (100%) 1 5 5Healthy 52  0 (0%) <0.00001 52 12 HD 6  0 (0%) <0.0001 2 6 AD 4  0 (0%)<0.0001 1 4 DLB 4  0 (0%) <0.0001 4 0 PSP 3  0 (0%) 0.0005 3 2 CBD 2  0(0%) 0.0059 2 2 FTD 3  0 (0%) 0.0005 3 0 Fisher Exact Test was used todetermine the P values compared to PD. The Miro1 intensities with DMSOand with CCCP were compared within the same subject in either ELISA orWestern by Mann-Whitney Test, and the numbers of the subjects with a P >0.05 or <0.05 were defined as “No. (Miro1 DMSO v.s. CCCP P > 0.05 or<0.05)”.

Notably, we discovered a unifying impairment in removing Miro1 from themitochondrial fractions at 6 hrs after CCCP treatment in 69 PD and atrisk lines (93.2%). By contrast, Miro1 was efficiently removed followingdepolarization in every single control subject (0%) (Table A). Thephenotype was more strikingly demonstrated when we imported the meanband intensities into a heat map where a lack of color change aftertreatment reflects the failure to remove Miro1 (FIG. 1D). The lack ofcolor change of Miro1 occurred broadly in PD subjects. A smallerfraction of PD cell lines also failed to remove Mitofusin2 after CCCPtreatment (FIG. 1D). Basal protein levels of Miro1 and Mitofusin2 werelargely comparable among all lines (FIG. 1D; P>0.0906).

This phenotype in Miro1 removal was significantly associated with PD(P<0.00001). The ratio of Miro1 intensity (with CCCP/with DMSO) was alsosignificantly correlated with PD (P<0.0001; FIG. 2A), but not with age(at sampling and onset) or gender (FIG. 2B-2D). There was no significantcorrelation between the Miro1 ratio and the disease progression (yearswith PD) or the clinical manifestations in subjects with the UnifiedParkinson's Disease Rating Scale (UPDRS), Hoehn and Yahr Scale, orMini-Mental Status Examination (FIG. 2E-2H). We confirmed that the cellpassaging numbers within the range of 5-19 had no influence on thephenotype. Taken together, these observations show that the failure toremove Miro1 from damaged mitochondria is a common cellular defect in alarge population of PD subjects.

The LRRK2 and PINK-Parkin Pathways Are Broadly Affected in PDFibroblasts. We have previously identified two parallel molecularpathways, both of which are essential for removing Miro from the OMM ofdepolarized mitochondria—LRRK2 and the PINK1-Parkin axis. To investigatethe mechanisms underlying Miro1 accumulation on damaged mitochondria inPD fibroblasts, we tested the hypothesis that the buildup was due to theimpairments of the LRRK2 or PINK1-Parkin pathway. We have establishedthat in wild-type control fibroblasts mitochondrial depolarization byCCCP treatment for only 1 hr triggers the recruitment of cytosolic LRRK2and Parkin to mitochondria and Miro1, prior to Miro1 removal at 6 hrs(FIG. 1A, 1C). Antibodies against LRRK2, Parkin, and Miro1 have beenvalidated in human cells lacking the corresponding genes.

We used this readout to screen all 96 fibroblast lines, and found avariety of phenotypes in subjects' cells. Some cell lines were impairedonly in recruiting LRRK2 to depolarized mitochondria, some impaired onlyin recruiting Parkin, and some cell lines were defective in recruitingboth proteins. Because the failure to relocate LRRK2 to damagedmitochondria disrupts the following removal of Miro1 but not ofMitofusin2, this result may explain the lower frequency of theMitofusin2 phenotype than Miro1 in those subjects (FIG. 1D). Wediscovered that in 7 PD cell lines the recruitment of both LRRK2 andParkin to damaged mitochondria appeared as normal when compared to thecontrols, while Miro1 removal was still compromised (FIG. 1D),suggesting that additional mechanisms might be at play in those cells.Basal levels of LRRK2 and Parkin were comparable in all lines(P>0.8684). Collectively, our results provide evidence that the LRRK2and PINK1-Parkin pathways are largely affected in PD subjects'fibroblasts, leading to a convergent downstream failure to remove Miro1from damaged mitochondria.

ELISA Confirms the High Frequency and Specificity of the Miro1 Phenotypefor PD Fibroblasts. We established an enzyme-linked immunosorbent assay(ELISA) to detect Miro1 response to CCCP (FIG. 3) which is useful forclinical laboratory use. We examined 14 PD/risk and 15 control celllines used in FIG. 1D. For each individual line, the result of Miro1response to mitochondrial depolarization (FIG. 3A) was consistent withthat from using mitochondrial fractionation and Western blotting (FIG.1D, Table S1A). We used this ELISA to validate additional independentcohorts. We included 40 healthy controls and 12 PD subjects from theStanford Alzheimer's Disease Research Center (ADRC) and the CoriellInstitute (Table S1B). We discovered that Miro1 was efficiently degradedupon depolarization in all control lines, but in none of the PD subjects(FIG. 1E); this finding verified the reliability of the high frequencyof Miro1 accumulation on depolarized mitochondria in the PD population(FIG. 1, Table A). We also obtained cell lines from subjects withmovement disorders that exhibit clinical presentations similar to PD,including 4 of sporadic Dementia with Lewy Bodies (DLB; Stanford), 3 ofFrontotemporal Degeneration (FTD; Coriell and NINDS), 2 of sporadicCorticobasal Degeneration (CBD; Mayo Clinic), and 3 of sporadicProgressive Supranuclear Palsy (PSP; Mayo Clinic) (Table S1B). Miro1 waseffectively degraded after CCCP treatment in all lines (FIG. 1F),demonstrating the specificity of Miro1 accumulation for PD. Theestablishment of the ELISA to detect Miro1 could facilitate clinicalapplications of our discovery.

Example 3 Effect of Miro-Reducing Agent on Mitochondrial Motility

Dopaminergic neurons expressing LRRK2G2019 are grown on glass coverslipsand are placed in a 35-mm petridish containing the Hibernate Elow-fluorescence medium (BrainBits) on a heated stage of 37° C., and areimaged with a 63×/N.A.0.9 water-immersion objective with excitation at561 nm or 488 nm. Neurons expressing LRRK2GS2019 are split into a testpool and a control pool of neurons. The test pool is treated with aMiro-reducing agent identified in Example 1, while the control pool isnot. After 10 hours, both pools of neurons are treated with FCCP for 14hours. mitochondrial motility is measured.

Time-lapse movies are obtained continually with 3-5 sec intervals beforeand after Miro-reducing agent is added (10 μM) is added. Axons longerthan 50 μm are selected for recording. Movies are recorded from 120 to300 min. 250 nM TMRM is applied for 30 min when needed. Forquantification, kymographs are generated from the time-lapse movies byImageJ, representing a 100-sec period either right before, or followingdifferent time points after the addition of Antimycin A. Each kymographis then imported into a macro written in Labview (NI, TX), andindividual mito-dsRed puncta are traced using a mouse-driven cursor atthe center of the mito-dsRed object. Using Matlab (The MathWorks, MA),the following parameters are quantified: 1) the instantaneous velocityof each mitochondrion, 2) the average velocity of those mitochondriathat are in motion, 3) the percent of time each mitochondrion is inmotion, 4) stop frequency, and 5) turn back frequency. The length andintensity of mitochondria are measured using ImageJ. Neurons in the testpool with depolarized mitochondria show reduced mitochondrial motilityas measured by the above parameters, whereas the control pool of neuronswith depolarized mitochondria show comparable mitochondrial motility asprior to addition of Miro-reducing agent.

Example 4 Effect of Miro-Reducing Agent on Parkinson's Disease in iPSCNeurons

LRRK2G2019S iPSC-derived neuronal cultures are treated with amitochondrial stressor Antimycin A with a series of concentrations for 6hrs to induce oxidative stress. LRRK2G2019S iPSC-derived neuronalcultures show an Antimycin A dose-dependent loss of neurons, depicting avulnerability to mitochondrial stress. LRRK2G2019S neurons treated withMiro-reducing agent rescued the loss of LRRK2G2019S neurons caused by 1or 10 μM Antimycin A, indicating a protection against neuronal losscaused by mitochondrial stress.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

TABLE 1A % Miro1 Molecular Concentration decrease Known Name MW formulatested (μM) in screen target class Description Temozolomide 194.15C₆H₆N₆O₂ 10 A DNA chemotherapy treatment of anaplastic astrocytoma,glioblastoma multiforme Escitalopram 324.39 C₂₀H₂₁FN₂O 10 A Transporterselective serotonin Oxalate reuptake inhibitor Benidipine HCl 505.56C₂₈H₃₁N₃O₆ 10 A Ion-channel calcium channel blockers Glycopyrrolate318.43 C₁₉H₂₈NO₃₊ 10 B GPCR long-acting muscarinic antagonistPhysostigmine 275.35 C₁₅H₂₁N₃O₂ 10 B Enzyme acetylcholinesteraseinhibitors Tranilast 327.33 C₁₈H₁₇NO₅ 10 B Enzyme antiasthmatics A =Reduction of Miro levels by >30%; B = Reduction of Miro levels by >20%and less than 30%; C = Reduction of Miro by >10% and less than 20%; D =Reduction of Miro by <10%

TABLE 1B Additional small molecules reducing Miro1 protein levelsMolecular Concentration % Miro1 decrease Known target Name formulatested (μM) in screen class Description Pazufloxacin C₁₆H₁₅FN₂O₄ 10 BUnknown antibacterials, nalidixic acid derivatives FlurbiprofenC₁₅H₁₃FO₂ 10 B Enzyme anti-inflammatory agents, ibuprofen derivativesLoxoprofen C₁₅H₁₈O₃ 10 B Enzyme anti-inflammatory agents, ibuprofenderivatives Omeprazole C₁₇H₁₉N₃O₃S 10 C Transporter antiulcer,benzimidazole derivatives A = Reduction of Miro levels by >30%; B =Reduction of Miro levels by >20% and less than 30%; C = Reduction ofMiro by >10% and less than 20%; D = Reduction of Miro by <10%

What is claimed is:
 1. A method to screen candidate agents for activityto reduce MIRO1 level, the method comprising: (a) contacting a candidateagent and a sample from a subject deficient in or suspected of beingdeficient in the removal of MIRO1, (b) measuring the MIRO1 level in thesample, (c) comparing the MIRO1 level in a sample with a control MIRO1level, and (d) assessing the activity of the candidate agent if theMIRO1 level in the sample is lower than the control MIRO1 level.
 2. Themethod of claim 1, wherein the sample comprises a cell.
 3. The method ofclaim 1 or 2, wherein the sample comprises a skin cell.
 4. The method ofany one of claims 1 to 3, wherein the sample with a control MIRO1 levelis from a healthy subject.
 5. The method of any one of claims 1 to 4,wherein the assessing comprises biochemical assays, western blotting orELISA.
 6. The method of any one of claims 1 to 5, wherein the MIRO1level is lower by about 20% or more than the control MIRO1 level.
 7. Themethod of any one of claims 1 to 6, wherein the MIRO1 level is lower byabout 30% or more than the control MIRO1 level.
 8. A method to determinethe MIRO1 status of a subject comprising measuring the Miro1 response tomitochondrial depolarization using biochemical assays, western blottingor ELISA, to determine if a subject is deficient in the removal of MIRO1following depolarization, wherein a subject deficient in the removal ofMIRO1 following depolarization is selected for treatment byadministration of a MIRO1 reducer.
 9. The method of claim 8, comprisingdetecting MIRO1 level in the subject, and comparing the MIRO1 level to acontrol MIRO1 level from a control subject.
 10. The method of claim 8 or9, wherein the detecting MIRO1 level comprises ELISA.
 11. The method ofany one of claims 8 to 10, wherein the detecting MIRO1 level comprisesdetecting MIRO1 in a sample in the subject.
 12. The method of claim 11,wherein the sample comprises a skin cell.
 13. The method of any one ofclaims 8 to 12, wherein the MIRO1 level in the subject is higher thanthe control MIRO1 level from a control subject.
 14. The method of anyone of claims 8 to 13, wherein the MIRO1 level in the subject is about20% or more than the control MIRO1 level from a control subject.
 15. Themethod of any one of claims 8 to 14, wherein the MIRO1 level in thesubject is about 30% or more than the control MIRO1 level from a controlsubject.
 16. The method of any one of claims 13 to 15, comprisingdiagnosing the subject with a neurodegenerative disorder.
 17. The methodof claim 16, wherein the neurodegenerative disorder is Parkinson'sdisease.
 18. The method of any one of claims 8 to 17, further comprisingtreating the subject with the MIRO1 reducer.
 19. The method of claim 18,further comprising monitoring MIRO1 levels before and after treatmentwith the MIRO1 reducer.
 20. A method of treating a neurodegenerativedisorder comprising administering to a subject in need thereof aMiro-reducing agent, wherein the Miro-reducing agent reduces Miro insporadic Parkinson's disease cells with depolarized mitochondria bythree standard deviations or more as compared to reduction of Miro incontrol depolarized sporadic Parkinson's disease cells with depolarizedmitochondria not contacted with the Miro-reducing agent.
 21. The methodof claim 20, wherein the healthy cells with depolarized mitochondria arehealthy cells contacted with a mitochondrial depolarizing agent.
 22. Amethod of treating a neurodegenerative disorder comprising administeringto a subject in need thereof a Miro-reducing agent, wherein theMiro-reducing agent reduces Miro in Parkinson's disease cells withdepolarized mitochondria by three standard deviations or more ascompared to reduction of Miro in control depolarized sporadicParkinson's disease cells with depolarized mitochondria not contactedwith the Miro-reducing agent.
 23. A method of treating aneurodegenerative disorder comprising administering to a subject in needthereof a Miro-reducing agent, wherein: (a) the Miro-reducing agentreduces Miro in Parkinson's disease cells with depolarized mitochondriaby two standard deviations or more as compared to reduction of Miro incontrol Parkinson's disease cells with depolarized mitochondria notcontacted with the Miro-reducing agent; and (b) the Miro 1 reducingagent reduces Miro in Parkinson's disease cells with non-depolarizedmitochondria by one standard deviation or less as compared to reductionof Miro in control Parkinson's disease cells with non-depolarizedmitochondria not contacted with the Miro-reducing agent.
 24. The methodof any one of claims 20 to 24, wherein the Parkinson's disease cellswith depolarized mitochondria are Parkinson's disease cells contactedwith a mitochondrial depolarizing agent.
 25. The method of any one ofclaims 20 to 25, wherein the Parkinson's disease cells are sporadicParkinson's disease cells.
 26. The method of any one of claims 20 to 26,wherein the Parkinson's disease cells are fibroblasts.
 27. A method oftreating a neurodegenerative disorder comprising administering to asubject in need thereof a Miro-reducing agent, wherein the Miro-reducingagent reduces Miro in the following assay: (a) fibroblast cells from asporadic Parkinson's disease patient are plated into wells of an array;(b) 24 hours after step (a), a candidate agent is added to a test welland the candidate agent is not added to a control well; (c) 10 hoursafter step (b), FCCP is added to the test well and control well; (d) 14hours after step (c) cells of the test well and control well are fixedwith ice-cold 90% methanol; and (e) the cells of the test well andcontrol well are immunostained with anti-Miro and4′,6-diamidino-2-phenylindole, dihydrochloride (DAPI) and imaged with aconfocal microscope to measure Miro intensity/cell for those images ofthe test well and control well; wherein when the candidate agent reducesMiro in the test well by three standard deviations or more relative tothe control well, the candidate agent is a Miro-reducing agent.
 28. Amethod of treating a neurodegenerative disorder comprising administeringto a subject in need thereof a Miro-reducing agent, wherein theMiro-reducing agent reduces Miro in sporadic Parkinson's disease cellsaccording to the following assay: (a) fibroblast cells from a sporadicParkinson's disease patient are plated into wells of an array; (b) 24hours after step (a), a candidate agent is added to a first test welland the candidate agent is not added to a first control well; (c) 10hours after step (b), FCCP is added to a first test well and firstcontrol well; (d) 14 hours after step (c), cells of the first test welland first control well are fixed with ice-cold 90% methanol; and (e) thecells of the first test well and first control well are immunostainedwith anti-Miro and 4′,6-diamidino-2-phenylindole, dihydrochloride (DAPI)and imaged with a confocal microscope to measure Miro intensity/cell forthose images of the first test well and first control well; and whereinthe Miro-reducing agent does not reduce Miro in sporadic Parkinson'sdisease cells according to the following assay: (a1) fibroblast cellsfrom a sporadic Parkinson's disease patient are plated into wells of anarray; (b1) 24 hours after step (a1), the candidate agent is added to asecond test well and is not added to a second control well; (c1) 14hours after step (b1), cells of the second test well and second controlwell are fixed with ice-cold 90% methanol; and (d1) the cells of thesecond test well and second control well are immunostained withanti-Miro and 4′,6-diamidino-2-phenylindole, dihydrochloride (DAPI) andimaged with a confocal microscope to measure Miro intensity/cell forthose images of the second test well and second control well; whereinwhen the candidate agent reduces Miro in the first test well by twostandard deviations or more relative to the first control well, and thecandidate agent reduces Miro in the second test well by less than onestandard deviation relative to the second control well, the candidateagent is a Miro-reducing agent.
 29. The method of any one of claims 20to 28, wherein the Miro-reducing agent reduces intracellular calciumlevels by 20% or more.
 30. The method of any one of claims 20 to 29,wherein the Miro-reducing agent has a molecular weight of from 100 to2000 Daltons.
 31. The method of any one of claims 20 to 30, wherein theMiro-reducing agent is an antibody, peptide, or protein or portion ofone thereof.
 32. The method of any one of claims 20 to 31, wherein theMiro-reducing agent binds to an EF hand protein.
 33. The method of anyone of claims 20 to 31, wherein the Miro-reducing agent is a calciumchannel blocker.
 34. The method of claim 33, wherein the calcium channelblocker is an L-type, and N-type calcium channel blocker.
 35. The methodof any one of claims 20 to 34, wherein the subject has elevated Miro.36. The method of any one of claims 20 to 35, wherein the subject has atleast about 20% or more, or about 30% or more MIRO1 than a controlsubject.
 37. The method of any one of claims 20 to 36, wherein thesubject is otherwise asymptomatic for the neurodegenerative disorder.38. The method of any one of claims 20 to 37, wherein theneurodegenerative disorder is Parkinson's disease.
 39. A method forselecting a subject for treatment with a therapeutic agent for aneurodegenerative disorder, comprising: (a) collecting cells from thesubject and evaluating a first control portion of the cells for thepre-depolarization Miro level in the cells; (b) contacting a second testportion of the cells with a depolarizing agent; and (c) evaluating thepost-depolarization Miro level in the second test portion of cellscontacted with the depolarizing agent and comparing the Miro level tothe pre-depolarization Miro level in the first control portion of cells;wherein when the post-depolarization Miro level in the second testportion of cells is reduced by 40% or less relative to thepre-depolarization Miro level in the first control portion of cells, thesubject is treated with a therapeutic agent for neurodegenerativedisorder.
 40. The method of claim 39, wherein when thepost-depolarization Miro level in the second test portion of cells isreduced by 10% to 50% relative to the pre-depolarization Miro level inthe first control portion of cells, the subject is treated with atherapeutic agent for neurodegenerative disorder.
 41. The method ofclaim 39 or 40, wherein the neurodegenerative disorder is Parkinson'sdisease.
 42. The method of any one of claims 39 to 41, wherein thesubject is asymptomatic for Parkinson's disease.
 43. The method of anyone of claims 39 to 42, wherein the therapeutic agent is selected fromlevodopa and dopamine antagonists.
 44. The method of any one of claims39 to 43, wherein the therapeutic agent is a Miro-reducing agent. 45.The method of any one of claims 39 to 44, wherein the therapeutic agenthas a molecular weight of from 100 to 2000 Daltons.
 46. The method ofany one of claims 39 to 44, wherein the therapeutic agent is anantibody, peptide, or protein or portion of one thereof.
 47. The methodof any one of claims 39 to 46, wherein the therapeutic agent binds to anEF hand protein.
 48. The method of any one of claims 39 to 47, whereinthe therapeutic agent is a calcium channel blocker.
 49. The method ofclaim 48, wherein the calcium channel blocker is an L-type and N-typecalcium channel blocker.
 50. A method of improving a condition of aging,comprising administering to a subject in need thereof a Miro-reducingagent.
 51. The method of claim 50, wherein the condition of aging isselected from memory impairment, muscle degeneration, arthritis,cardiovascular disease, osteoporosis, glaucoma, dementia, maculardegeneration, and cataract.
 52. The method of claim 50 or 51, whereinthe Miro-reducing agent is administered prophylactically.